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Kukho imfuneko yenkqubo ethembekileyo ye-in vitro enokuvelisa ngokuchanekileyo indawo ye-physiological yentliziyo yokuvavanya iziyobisi.Ukufumaneka okulinganiselweyo kweenkqubo zenkcubeko yezicubu zentliziyo yomntu kuye kwakhokelela ekutolikweni okungachanekanga kwemiphumo yeziyobisi zentliziyo.Apha, siye saphuhlisa imodeli yenkcubeko ye-cardiac tissue (CTCM) eyenza i-electromechanically ivuselele izilayi zentliziyo kwaye iqhube i-physiological stretch ngexesha le-systolic kunye ne-diastolic yezigaba zomjikelezo wenhliziyo.Emva kweentsuku ezili-12 zenkcubeko, le ndlela iphucule ngokuyinxenye ukusebenza kwamacandelo entliziyo, kodwa ayizange igcine ngokupheleleyo imfezeko yolwakhiwo.Ngoko ke, emva kokuhlolwa kwe-molecule encinci, sifumene ukuba ukongezwa kwe-100 nM triiodothyronine (T3) kunye ne-1 μM i-dexamethasone (i-Dex) kwi-medium yethu igcine i-microstructure yamacandelo kwiintsuku ze-12.Ngokudibanisa nonyango lwe-T3 / Dex, inkqubo ye-CTCM igcine iiprofayili zokubhaliweyo, ukusebenza, umsebenzi we-metabolic, kunye nokunyaniseka kwesakhiwo kwinqanaba elifanayo njengezicubu zentliziyo entsha kwiintsuku ze-12.Ukongezelela, ukugqithiswa ngokugqithiseleyo kwezicubu zentliziyo kwinkcubeko kubangela ukubonakaliswa kwe-hypertrophic cardiac, ukubonelela ubungqina bokukwazi kwe-CTCM ukulinganisa iimeko ze-hypertrophic ezibangelwa yi-cardiac stretch.Ukuqukumbela, i-CTCM inokumisela i-physiology kunye ne-pathophysiology yentliziyo kwinkcubeko ixesha elide, ivumela ukuhlolwa kweziyobisi okuthembekileyo.
Ngaphambi kophando lweklinikhi, iinkqubo ezithembekileyo ze-vitro ziyafuneka ezinokuphinda zivelise ngokuchanekileyo indawo ye-physiological yentliziyo yomntu.Iinkqubo ezinjalo kufuneka zilinganise ukunwebeka okutshintshileyo komatshini, izinga lokubetha kwentliziyo, kunye neempawu ze-electrophysiological.Iimodeli zezilwanyana zisetyenziswa ngokuqhelekileyo njengeqonga lokuhlola i-cardiac physiology enokuthembeka okulinganiselweyo ekuboniseni imiphumo yeziyobisi entliziyweni yomntu1,2.Ekugqibeleni, i-Ideal Cardiac Tissue Culture Experimental Model (CTCM) ngumzekelo onomdla kakhulu kwaye uchanekileyo kwiindlela ezahlukeneyo zonyango kunye ne-pharmacological interventions, ukuvelisa ngokuchanekileyo i-physiology kunye ne-pathophysiology yentliziyo yomntu3.Ukungabikho kwenkqubo enjalo kunciphisa ukufunyanwa konyango olutsha lokusilela kwentliziyo4,5 kwaye kukhokelele kwi-cardiotoxicity yeziyobisi njengesizathu esikhulu sokuphuma kwimarike6.
Kule minyaka ilishumi idlulileyo, amachiza asibhozo angengawo awentliziyo arhoxisiwe kusetyenziso lweklinikhi kuba abangela ukolulwa kwexesha le-QT elikhokelela kwi-ventricular arrhythmias kunye nokufa ngequbuliso7.Ke ngoko, kukho imfuneko eyandayo yezicwangciso ezithembekileyo zokuhlola kwangaphambili ukuvavanya ukusebenza kwentliziyo kunye netyhefu.Ukusetyenziswa kwamva nje kwe-pluripotent stem cell-derived cardiomyocytes (i-hiPS-CM) ekuhlolweni kweziyobisi kunye nokuvavanya ubuthi kunika isisombululo esincinci kule ngxaki.Nangona kunjalo, ubume be-hiPS-CMs kunye nokungabikho kobunzima be-multicellular yezicubu zenhliziyo yimida emikhulu yale ndlela.Uphononongo lwakutsha nje lubonise ukuba lo mda unokoyiswa ngokuyinxenye ngokusebenzisa i-hiPS-CM yokuqala ukwenza ii-hydrogel zezicubu zentliziyo kamsinya nje emva kokuqalisa kokuqhekeka okuzenzekelayo kwaye ngokuthe ngcembe ukwandisa ukuvuselela kombane ngokuhamba kwexesha.Nangona kunjalo, ezi microtissues ze-hiPS-CM azinayo i-electrophysiological eqolileyo kunye neepropathi zekhontrakthi ze-myocardium yabantu abadala.Ukongeza, izicubu zentliziyo yomntu zinesakhiwo esinzima ngakumbi, esibandakanya umxube ohlukeneyo weentlobo ezahlukeneyo zeeseli, kubandakanya iiseli ze-endothelial, i-neurons, kunye ne-stromal fibroblasts, ezidityaniswe ziiseti ezithile zeeproteni ze-matrix ezingaphandle.Le heterogeneity ye-non-cardiomyocyte populations11,12,13 kwintliziyo yabantu abadala abancancisayo ngumqobo omkhulu wokulinganisa izicubu zentliziyo usebenzisa iintlobo zeeseli zomntu ngamnye.Le mida ingundoqo igxininisa ukubaluleka kokuphuhlisa iindlela zokuhlakulela izicubu ze-myocardial ezingaguqukiyo phantsi kweemeko ze-physiological and pathological.
Amacandelo amancinci akhuliswe (300 µm) entliziyo yomntu angqineke eyimodeli ethembisayo yemyocardium yomntu.Le ndlela ibonelela ngokufikelela kwinkqubo epheleleyo ye-3D ye-multicellular efana nezicubu zentliziyo yomntu.Nangona kunjalo, kude kube ngu-2019, ukusetyenziswa kwamacandelo entliziyo akhulisiweyo kwanqunyulwa kukusinda kwenkcubeko emfutshane (24 h).Oku kubangelwa inani lezinto ezibandakanya ukungabikho kokwelula ngokomzimba, i-air-liquid interface, kunye nokusetyenziswa kweendaba ezilula ezingaxhasi iimfuno zezicubu zenhliziyo.Ngo-2019, amaqela ophando aliqela abonise ukuba ukubandakanya izinto ezisebenza ngoomatshini kwiinkqubo zenkcubeko yezicubu zentliziyo kunokwandisa ubomi benkcubeko, kuphuculwe ukubonakaliswa kwentliziyo, kunye nokulinganisa isifo sentliziyo.Izifundo ezimbini ezintle ze-17 kunye ne-18 zibonisa ukuba ukulayisha uniaxial ngoomatshini kunempembelelo entle kwi-phenotype yentliziyo ngexesha lenkcubeko.Nangona kunjalo, ezi zifundo azizange zisebenzise i-dynamic-dimensional physico-mechanical loading ye-cardiac cycle, ekubeni amacandelo entliziyo alayishwe kunye ne-isometric tensile forces 17 okanye i-auxotonic yomgca yokulayisha i-18.Ezi ndlela zokwelula izicubu zibangele ukunyanzelwa kweejene ezininzi zenhliziyo okanye ukugqithiswa kweejene ezihambelana neempendulo ezingaqhelekanga ezilula.Ngokukodwa, uPitoulis et al.I-19 iphuhlise indawo yokuhlambela yenkcubeko yesilayi sentliziyo eguqukayo yokwakhiwa kwakhona komjikelo wentliziyo kusetyenziswa impendulo ye-transducer enamandla kunye noxinzelelo.Nangona le nkqubo ivumela ukuchaneka ngakumbi kwi-vitro cardiac cycle modeling, ubunzima kunye ne-low throughput yendlela kunciphisa ukusetyenziswa kwale nkqubo.I-laboratory yethu isandul 'ukuphuhlisa inkqubo yenkcubeko elula isebenzisa ukuvuselela umbane kunye ne-medium optimized ukuze kugcinwe ukusebenza kwamacandelo e-porcine kunye nentliziyo yomntu ukuya kuthi ga kwiintsuku ze-620,21.
Kulo mbhalo wesandla wangoku, sichaza imodeli yenkcubeko ye-cardiac tissue (CTCM) sisebenzisa amacandelo entliziyo ye-porcine ebandakanya i-humoral cues ukubuyisela i-physiology ye-cardiac ye-three-dimensional kunye ne-pathophysiological distension ngexesha lomjikelezo wenhliziyo.Le CTCM inokunyusa ukuchaneka kokuqikelelwa kwangaphambili kweziyobisi ukuya kwinqanaba elingazange liphunyezwe ngokubonelela ngexabiso eliphantsi, inkqubo ye-cardiac ephakathi elinganisa i-physiology / pathophysiology yentliziyo ye-mammalian yokuvavanya iziyobisi zangaphambili.
Iimpawu ze-Hemodynamic mechanical zidlala indima ebalulekileyo ekugcineni umsebenzi we-cardiomyocyte kwi-vitro 22,23,24.Kulo mbhalo wesandla wangoku, siye saqulunqa i-CTCM (Umfanekiso 1a) onokulingisa indawo yenhliziyo yabantu abadala ngokubangela ukuvuselela umbane kunye nomatshini kwii-frequencies ze-physiological (1.2 Hz, i-72 beats ngomzuzu).Ukuze ugweme i-tissue eyongeziweyo yokwelula ngexesha le-diastole, isixhobo sokuprinta se-3D sisetyenziselwa ukwandisa ubungakanani bezicubu nge-25% (umzobo 1b).Ukunyanzeliswa kombane okubangelwa yinkqubo ye-C-PACE kumiselwe ixesha lokuqalisa i-100 ms ngaphambi kokuba i-systole isebenzisa inkqubo yokufumana idatha ukuvelisa ngokupheleleyo umjikelezo wenhliziyo.Inkqubo yenkcubeko ye-tissue isebenzisa i-actuator ye-pneumatic programmable (LB Engineering, eJamani) ukunyusa i-cyclically i-membrane ye-silicone eguquguqukayo ukuze kubangele ukwandiswa kweentliziyo zentliziyo kwigumbi eliphezulu.Inkqubo ixhunywe kumgca womoya wangaphandle ngokusebenzisa i-transducer yoxinzelelo, okwenza kube lula ukulungelelanisa ngokuchanekileyo uxinzelelo (± 1 mmHg) kunye nexesha (± 1 ms) (Umfanekiso 1c).
a Qhoboshela icandelo lethishu kwiringi yenkxaso eyi-7 mm, eboniswe ngebhlowu, ngaphakathi kwegumbi lenkcubeko yesixhobo.Igumbi lenkcubeko lihlukaniswe kwigumbi lomoya ngenwebu encinci ye-silicone ethambileyo.Beka i-gasket phakathi kwegumbi ngalinye ukukhusela ukuvuza.Isivalo sesixhobo sinee-electrode zegraphite ezibonelela ngovuselelo lombane.b Umelo olucwangcisiweyo lwesixhobo esikhulu sethishu, iringi yesikhokelo kunye neringi yenkxaso.Amacandelo eethishu (brown) abekwe kwisixhobo esinobukhulu obugqithisileyo kunye neringi yesikhokelo ebekwe kwigroove kumda ongaphandle wesixhobo.Ukusebenzisa isikhokelo, beka ngononophelo umsesane wenkxaso ogqunywe nge-tissue acrylic adhesive phezu kwecandelo le-cardiac tissue.c Igrafu ebonisa ixesha lokuvuselela umbane njengomsebenzi woxinzelelo lwegumbi lomoya elilawulwa yi-programmable pneumatic actuator (PPD).Isixhobo sokufumana idatha sisetyenziselwe ukuvumelanisa uvuselelo lombane kusetyenziswa izinzwa zoxinzelelo.Xa uxinzelelo kwigumbi lenkcubeko lifikelela kumda obekiweyo, isignali ye-pulse ithunyelwa kwi-C-PACE-EM ukuqalisa ukuvuselela umbane.d Umfanekiso wee-CTCM ezine ezibekwe kwishelufu yokufukamela.Izixhobo ezine zixhunywe kwi-PPD enye ngesekethe ye-pneumatic, kwaye ii-sensor zoxinzelelo zifakwe kwi-valve ye-hemostatic ukujonga uxinzelelo kwisiphaluka se-pneumatic.Isixhobo ngasinye sinamacandelo amathandathu eethishu.
Ukusebenzisa i-actuator eyodwa ye-pneumatic, sakwazi ukulawula izixhobo ze-CTCM ze-4, nganye yazo inokubamba amacandelo e-6 (umzobo 1d).Kwi-CTCM, uxinzelelo lomoya kwigumbi lomoya liguqulwa libe yingcinezelo ye-synchronous kwi-fluid room kwaye ibangele ukwandiswa kwe-physiological of heart slice (Umfanekiso we-2a kunye ne-Movie ye-Supplementary 1).Uvavanyo lokwelula kwezicubu kwi-80 mm Hg.Ubugcisa.ubonise ukunweba kwamacandelo e-tissue ngama-25% (umzobo 2b).Olu luhlu lwepesenti lubonakaliswe ukuba luhambelana nobude be-physiological sarcomere ye-2.2-2.3 µm ye-contractility yecandelo le-cardiac yesiqhelo17,19,25.Ukunyakaza kwezicubu kuhlolwe kusetyenziswa izicwangciso zekhamera yesiqhelo (uMzobo owongezelelweyo 1).I-amplitude kunye nesantya sokuhamba kwezicubu (Umfanekiso 2c, d) uhambelana nokwelula ngexesha lomjikelezo wenhliziyo kunye nexesha ngexesha le-systole kunye ne-diastole (umzobo 2b).Ukwelula kunye nesantya sezicubu zenhliziyo ngexesha lokuqhawula kunye nokuphumla kwahlala kuhleli iintsuku ze-12 kwinkcubeko (Umfanekiso 2f).Ukuvavanya umphumo wokuvuselela umbane kwi-contractility ngexesha lenkcubeko, senze indlela yokumisela ukukhubazeka okusebenzayo usebenzisa i-algorithm ye-shading (i-Supplementary Fig. 2a, b) kwaye sakwazi ukuhlukanisa phakathi kokukhubazeka kunye nangaphandle kokuvuselela umbane.Icandelo elifanayo lentliziyo (Umfanekiso 2f).Kwindawo ehambayo yokunqunyulwa (i-R6-9), i-voltage ngexesha lokuvuselela umbane lalingama-20% aphezulu kunokuba kungabikho ukuvuselela kombane, okubonisa igalelo lokuvuselela umbane kumsebenzi we-contractile.
Umkhondo omele uxinzelelo lwegumbi lomoya, uxinzelelo lwegumbi lolwelo, kunye nemilinganiselo yokunyakaza kwezicubu ziqinisekisa ukuba uxinzelelo lwegumbi lutshintsha uxinzelelo lwegumbi lolwelo, olubangela ukunyakaza okuhambelanayo kwesilayi sethishu.b Umkhondo omele ipesenti yolula (oluhlaza okwesibhakabhaka) yamacandelo e-tissue ahambelana nokwelula kwepesenti (orenji).c Intshukumo elinganisiweyo yesilayi senhliziyo ihambelana nesantya esilinganisiweyo sokunyakaza.(d) Iindlela ezimele intshukumo yebhayisikile (umgca oblowu) kunye nesantya (umgca wamachokoza orenji) kwisiqwenga sentliziyo.e Ubungakanani bexesha lomjikelo (n = 19 izilayi kwiqela ngalinye, ukusuka kwiihagu ezahlukeneyo), ixesha lokunqanda (n = 19 izilayi kwiqela ngalinye), ixesha lokuphumla (n = 19 izilayi kwiqela ngalinye, kwiihagu ezahlukeneyo), ukunyakaza kwezicubu (n = 25).izilayi)/iqela elisuka kwiihagu ezahlukeneyo), isantya esiphezulu sesystolic (n = 24(D0), 25(D12) izilayi/iqela elisuka kwiihagu ezahlukeneyo) kunye nepeak rate yokuphumla (n=24(D0), 25(D12) izilayi/iqela elisuka kwiihagu ezahlukeneyo).Uvavanyo loMfundi olunemisila emibini alubonisanga mahluko ubalulekileyo kuyo nayiphi na ipharamitha.f Uhlalutyo loxinzelelo olumeleyo lulandelela amacandelo ezicubu ezino (obomvu) nangaphandle (obhlowu) ukuvuselela umbane, iindawo ezilishumi zengingqi zamacandelo eethishu ukusuka kwicandelo elinye.Iipaneli ezisezantsi zibonisa ubungakanani bepesenteji yokwahlukana kuxinzelelo kumacandelo e-tissue kunye nangaphandle kokuvuselela kombane kwiindawo ezilishumi ezivela kumacandelo ahlukeneyo. (n = izilayi ezisi-8/iqela elivela kwiihagu ezahlukeneyo, kwenziwa uvavanyo lwe-t lomfundi olunemisila emibini; ****p <0.0001, **p <0.01, *p <0.05). (n = izilayi ezisi-8/iqela elivela kwiihagu ezahlukeneyo, kwenziwa uvavanyo lwe-t lomfundi olunemisila emibini; ****p <0.0001, **p <0.01, *p <0.05). (n = 8 срезов/группу от разных свиней, проводится двусторонний t-критерий Стьюдента; ****p<0,0001, **p<0,01, *p<0,05). (n = 8 amacandelo/iqela elivela kwiihagu ezahlukeneyo, uvavanyo lomfundi olunemisila emibini; ****p<0.0001, **p<0.01, *p<0.05). （n = 8 片/组，來自不同的猪，进行双尾学生t 检验；****p <0.0001,**p <0.01,*p <0.05）. （n = 8 片/组，來自不同的猪，进行双尾学生t 检验；****p <0.0001,**p <0.01,*p <0.05）. (n = 8 срезов/группу, от разных свиней, двусторонний критерий Стьюдента; ****p <0,0001, **p <0,01, *p <0,05). (n = 8 amacandelo/iqela, ukusuka kwiihagu ezahlukeneyo, uvavanyo lomfundi olunemisila emibini; ****p<0.0001, **p<0.01, *p<0.05).Iibha zeemposiso zibonisa intsingiselo ± ukutenxa okusemgangathweni.
Kwinkqubo yethu yangaphambili ye-static biomimetic heart slice yenkcubeko [20, 21], siye sagcina ukusebenza, ukusebenza, kunye nolwakhiwo lwemfezeko yezilayi zentliziyo kangangeentsuku ezi-6 ngokufaka uvuselelo lombane kunye nokwenza ngcono ukwakheka okuphakathi.Nangona kunjalo, emva kweentsuku ezili-10, la manani ehla kakhulu.Siza kubhekisela kumacandelo akhuliswe kwinkqubo yethu yangaphambili ye-static biomimetic culture 20, i-21 imiqathango yokulawula (Ctrl) kwaye siya kusebenzisa i-medium yethu yangaphambili ephuculweyo njengeemeko ze-MC kunye nenkcubeko phantsi kokuvuselelwa komatshini kunye nombane (CTCM).ebizwa .Okokuqala, sinqume ukuba ukuvuselela oomatshini ngaphandle kokuvuselela umbane kwakunganelanga ukugcina ukusebenza kwezicubu kwiintsuku ze-6 (i-Supplementary Fig. 3a, b).Kuyathakazelisa ukuba ngokusungulwa kwe-physio-mechanical kunye nokuvuselela umbane usebenzisa i-STCM, ukusebenza kwamacandelo entliziyo yeentsuku ze-12 kwahlala kufana namacandelo entliziyo entsha phantsi kweemeko ze-MS, kodwa kungekhona phantsi kweemeko ze-Ctrl, njengoko kuboniswe kuhlalutyo lwe-MTT (Umfanekiso 1).3a).Oku kuphakamisa ukuba ukuvuselela oomatshini kunye nokulinganisa umjikelezo wenhliziyo kunokugcina amacandelo e-tissue esebenzayo ngokuphindwe kabini njengoko kuchazwe kwinkqubo yethu yangaphambili yenkcubeko.Nangona kunjalo, uvavanyo lwengqibelelo yesakhiwo samacandelo e-tissue ngo-immunolabeling ye-cardiac troponin T kunye ne-connexin 43 ibonise ukuba i-connexin ye-43 ibonakaliso yayiphezulu kakhulu kwi-MC tissues ngosuku lwe-12 kunolawulo ngosuku olufanayo.Nangona kunjalo, i-uniform connexin inkcazo ye-43 kunye nokwakheka kwe-Z-disc ayizange igcinwe ngokupheleleyo (Umfanekiso 3b).Sisebenzisa i-Artificial Intelligence (AI) isakhelo ukulinganisa ingqibelelo yesakhiwo sethishu26, umbhobho wokufunda nzulu osekwe kumfanekiso osekwe kwi-troponin-T kunye ne-connexin staining43 ukulinganisa ngokuzenzekelayo imfezeko yesakhiwo kunye ne-fluorescence yezilayi zentliziyo ngokomelela kwendawo.Le ndlela isebenzisa iNethiwekhi ye-Convolutional Neural (CNN) kunye nesikhokelo sokufunda esinzulu ukulinganisa ngokuthembekileyo ukunyaniseka kwesakhiwo sezicubu zenhliziyo ngendlela ezenzekelayo kunye nokungakhethi, njengoko kuchazwe kwireferensi.I-26. Izicubu ze-MC zibonise ukufana kwesakhiwo esiphuculweyo kumhla we-0 xa kuthelekiswa namacandelo okulawula i-static.Ukongezelela, i-Masson's trichrome staining ibonise ipesenti ephantsi kakhulu ye-fibrosis phantsi kweemeko ze-MS xa kuthelekiswa neemeko zokulawula kwi-12 yenkcubeko (Umfanekiso 3c).Nangona i-CTCM yandisa ukusebenza kwamacandelo eethambo zentliziyo ngosuku lwe-12 ukuya kwinqanaba elifana nelo lithambo lentliziyo entsha, ayizange iphucule kakhulu ingqibelelo yesakhiwo samacandelo entliziyo.
Igrafu yeBar ibonisa ubungakanani beMTT yokwenzeka kwezilayi zentliziyo entsha (D0) okanye inkcubeko yamacwecwe entliziyo kangangeentsuku ezili-12 nokuba kwinkcubeko engatshintshiyo (D12 Ctrl) okanye kwiCTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) izilayi/iqela lenziweyo ukusuka kwi-1 ukuya kwi- # 0VA yovavanyo, # 0 ithelekiswa ne #0VA eyahlukileyo; 0 kunye ** p <0.01 xa kuthelekiswa ne-D12 Ctrl). igrafu yeBar ibonisa ubungakanani beMTT yokwenzeka kwezilayi zentliziyo entsha (D0) okanye inkcubeko yamacwecwe entliziyo kangangeentsuku ezili-12 nokuba kwinkcubeko engatshintshiyo (D12 Ctrl) okanye kwiCTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) izilayi/iqela le0/iqela le-0 kuthelekiswa ne- # 0VA yehagu; D0 kunye ** p <0.01 xa kuthelekiswa ne-D12 Ctrl).i-histogram ibonisa ubungakanani bokusebenza kwe-MTT yamacandelo entliziyo entsha (i-D0) okanye inkcubeko yamacandelo entliziyo kwiintsuku ze-12 nokuba yinkcubeko ye-static (D12 control) okanye i-CTCM (D12 MC) (n = 18 (D0), 15 (D12 control) )), i-12 (D12 MC) amacandelo / iqela lenziwa kwi-ANO-iqela le-ANO;####p < 0,0001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). ####p <0.0001 xa kuthelekiswa ne-D0 kunye ne-**p <0.01 xa kuthelekiswa ne-D12 Ctrl). a 条形图显示在静态培养(D12 CTRL) 或CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl) 新鲜心脏切片(D0) 或則片(D0) 18活力的量化)，來自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；与D0 相比，####p <0.0001, Ctrl* CT001, . a 条形图显示在静态培养(D12 Ctrl) 或CTCM (D12 MC) (n = 18 (D0), 15 (D12 CTRL) 新鲜心脏切片(D0) 中光 MC 片(D0) 2组，进行单向ANOVA 测试；与D0 相比，####p <0.0001，与D12 Ctrl 相比，**p.)i-histogram ebonisa ubungakanani be-MTT esebenzayo kumacandelo entliziyo entsha (i-D0) okanye amacandelo entliziyo akhuliswe iintsuku ze-12 kwinkcubeko ye-static (D12 control) okanye i-CTCM (D12 MC) (n = 18 (D0), i-15 (i-D12 control)), i-12 (i-D12 MC) amacandelo / iqela elivela kwi-ANOVA eyahlukileyo, uvavanyo olulodwa;####p < 0,0001 по сравнению с D0, **p < 0,01 по сравнению с D12 Ctrl). ####p <0.0001 xa kuthelekiswa ne-D0, **p <0.01 xa kuthelekiswa ne-D12 Ctrl).b I-Troponin-T (eluhlaza), i-connexin 43 (obomvu) kunye ne-DAPI (eluhlaza okwesibhakabhaka) kumacandelo entliziyo asanda yedwa (D0) okanye amacandelo entliziyo akhuliswe phantsi kweemeko ze-static (Ctrl) okanye iimeko ze-CTCM (MC) iintsuku ze-12) zemifanekiso emele i-immunofluorescence (isikali esingenanto = 100 µm). I-Artificial intelligence quantification of the heart tissue ofstructural integrity (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izilayi / iqela ngalinye ukusuka kwihagu eyahlukileyo, uvavanyo lwe-ANOVA lwendlela enye lwenziwa; I-Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izilayi / iqela ngalinye kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA lwendlela enye lwenziwa; Количественная оценка структурной целостности сердечной ткани искусственным интеллектом (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) срезов/ ся однофакторный тест ANOVA; ####p < 0,0001 по сравнению с D0 и ****p < 0,0001 по сравнению с D12 Ctrl). Ubungakanani bengqibelelo yesakhiwo sezicubu zentliziyo ngobukrelekrele bokwenziwa (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) amacandelo/amaqela asuka kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA lwendlela enye lwenziwa;人工智能量化心脏组织结构完整性（n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izilayi/iqela ngalinye lehagu eyahlukileyo, indlela enye ANOVA uvavanyo;##0#000 **** **** 0001 与D12 Ctrl 相比）.人工智能量化心脏组织结构完整性（n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izilayi/iqela nganye yeehagu ezahlukeneyo, indlela-nye yovavanyo lwe-ANOVA;##0#000p **** ****. 001 与D12 Ctrl 相比）. Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC/MC) сзрка ней, односторонний тест ANOVA; ####p <0,0001 vs. D0 Для сравнения ****p < 0,0001 по сравнению с D12 Ctrl). Ubukrelekrele bokwenziwa ukulinganisa ingqibelelo yesakhiwo sezicubu zentliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) amacandelo / iqela ngalinye leehagu ezahlukeneyo, uvavanyo lwe-ANOVA lwendlela enye; c Imifanekiso emele (ekhohlo) kunye nobungakanani (ekunene) kwizilayi zentliziyo ezidyojwe ngebala le-trichrome lika-Masson (Isikali esingenanto = 500 µm) (n = 10 izilayi/iqela ngalinye ukusuka kwihagu eyahlukileyo, uvavanyo lwe-ANOVA lwendlela enye luyenziwa; ####p <0.0001 xa kuthelekiswa ne-D00 kunye ne-Dtrl 1 xa kuthelekiswa ne-D00 kunye ne-D*10 ***p). c Imifanekiso emele (ekhohlo) kunye nobungakanani (ekunene) kwizilayi zentliziyo ezidyojwe ngebala le-trichrome lika-Masson (Isikali esingenanto = 500 µm) (n = 10 izilayi/iqela ngalinye ukusuka kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA lwendlela enye luyenziwa; #### p <0.0001 xa kuthelekiswa ne-D00 kunye ne-Dtrl 1 xa kuthelekiswa ne-D00 kunye ne-D*10 ***p). c Репрезентативные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красителем Масязом50 (n = 10 срезов/группу от разных свиней, выполняется односторонний тест ANOVA; #### p <0,0001 по сравнению с D0 kunye ***p <0,0001                               c Imifanekiso emele (ekhohlo) kunye nomlinganiselo (ekunene) wamacandelo entliziyo adityaniswe ngebala le-trichrome lika-Masson (isikali esingafakwanga = 500 µm) (n = 10 amacandelo/iqela elivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA lwendlela enye lwenziwe; #### p <0 .0001 xa kuthelekiswa ne-D0 kunye ne-Ctrl 1 *20 <p. c 用Masson 三色染料染色的心脏切片的代表性图像（左）和量化（右）（裸尺度= 500 ）尺度= 500 ）尺度=500组，每组來自不同的猪，进行单向ANOVA 测试；#### p <0.0001 与D0 相比，***p <0.001 与D12 Ctrl 相比。 C 用 mason 三 色 染料的 心脏 切片 的 代表性 （左 左） 量化 （右） 裸尺度 裸尺度 裸尺庰 裸尺度 裸尺庰 裸尺度 裸尺庰 裸尺庰 裸尺度 0 µm） （n = 10 个 切片 组 每 來自 不同 猪 , 进行 单向 单向 Anova 测试；#### p <0.00001 Xhuma i-D12 Ctrl 相比）. c Репрезентативные изображения (слева) и количественный анализ (справа) срезов сердца, окрашенных трихромным красителем Массом 1 =0 срезов/группа, каждый от другой свиньи, протестировано с помощью однофакторного дисперсионного анализа Ctrl). c Imifanekiso emele (ekhohlo) kunye nobungakanani (ngasekunene) bamacandelo entliziyo adyojwe ngebala le-trichrome lika-Masson (elingenanto = 500 µm) (n = 10 amacandelo/iqela, ngalinye lisuka kwihagu eyahlukileyo, livavanywe ngokuhlalutya kwendlela enye yomahluko ;### # p <0.0001 <0.0001 xa kuthelekiswa no-D010,*20 Ctrl xa kuthelekiswa ne-D010,*20).Iibha zeemposiso zibonisa intsingiselo ± ukutenxa okusemgangathweni.
Sasicinga ukuba ngokongeza iamolekyu ezincinci kwinkcubeko yenkcubeko, ingqibelelo ye-cardiomyocyte inokuphuculwa kwaye ukuphuhliswa kwe-fibrosis kuncitshiswe ngexesha lenkcubeko ye-CTCM.Ngoko ke sihlolisise iimolekyuli ezincinci sisebenzisa iinkcubeko zethu zokulawula i-static20,21 ngenxa yenani elincinci lezinto eziphazamisayo.I-Dexamethasone (Dex), i-triiodothyronine (T3), kunye ne-SB431542 (SB) zikhethwe kwesi sikrini.Ezi molekyuli ezincinci ziye zasetyenziswa ngaphambili kwiinkcubeko ze-hiPSC-CM ukukhuthaza ukuvuthwa kwe-cardiomyocytes ngokunyusa ubude be-sarcomere, i-T-tubules, kunye nesantya sokuqhuba.Ukongezelela, zombini i-Dex (i-glucocorticoid) kunye ne-SB ziyaziwa ngokucinezela ukuvuvukala29,30.Ngoko ke, sivavanye ukuba ukufakwa kwenye okanye ukudibanisa kwezi molekyuli ezincinci kuya kuphucula ukunyaniseka kwesakhiwo samacandelo entliziyo.Ukuhlolwa kokuqala, i-dose ye-compound nganye ikhethwe ngokusekelwe kugxininiso oluqhelekileyo olusetyenziswa kwiimodeli zenkcubeko yeseli (1 μM Dex27, 100 nM T327, kunye ne-2.5 μM SB31).Emva kweentsuku ze-12 zenkcubeko, ukudibanisa kwe-T3 kunye ne-Dex kubangele ukunyaniseka kwesakhiwo se-cardiomyocyte kunye nokulungiswa okuncinci kwe-fibrous (i-Supplementary Figures 4 kunye ne-5).Ukongeza, ukusetyenziswa kokuphindwe kabini okanye kabini oku kugxininiswa kwe-T3 kunye ne-Dex kuvelise iziphumo ezinobungozi xa kuthelekiswa nokugxininiswa okuqhelekileyo (i-Supplementary Fig. 6a,b).
Emva kokuhlolwa kokuqala, senze uthelekiso lwentloko-entloko yeemeko zenkcubeko ye-4 (Umfanekiso 4a): I-Ctrl: amacandelo entliziyo akhuliswe kwinkcubeko yethu echazwe ngaphambili echazwe kusetyenziswa i-medium yethu ephuculweyo;20.21 TD: T3 kunye neCtrl s Added Dex ngoLwesithathu;I-MC: amacandelo entliziyo akhuliswe kwi-CTCM kusetyenziswa indawo yethu elungiselelwe ngaphambili;kunye ne-MT: I-CTCM ene-T3 kunye ne-Dex eyongezwe phakathi.Emva kweentsuku ze-12 zokulima, ukusebenza kwe-MS kunye ne-MT izicubu zahlala zifana nezicubu ezitsha ezihlolwe yi-MTT assay (Fig. 4b).Kuyathakazelisa ukuba ukongezwa kwe-T3 kunye ne-Dex kwiinkcubeko ze-transwell (TD) akuzange kubangele ukuphuculwa okuphawulekayo ekusebenzeni xa kuthelekiswa neemeko ze-Ctrl, ebonisa indima ebalulekileyo yokuvuselela oomatshini ekugcineni ukusebenza kwamacandelo entliziyo.
umzobo woyilo loMfuniselo obonisa iimeko ezine zenkcubeko ezisetyenziselwa ukuvavanya imiphumo yokuvuselela oomatshini kunye ne-T3 / Dex supplementation phakathi kweentsuku ze-12. b Igrafu yeBar ibonisa ubungakanani bokusebenza kweentsuku ze-12 inkcubeko yeposi kuzo zonke iimeko ze-4 zenkcubeko (Ctrl, TD, MC, kunye ne-MT) xa kuthelekiswa nezilayi zentliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kunye ne-D12 MT), 12 (D12 MC) izilayi / iqela le-0 , # ANOVA, uvavanyo olulodwa 0; ###p <0.001 xa kuthelekiswa ne-D0 kunye ne-**p <0.01 xa kuthelekiswa ne-D12 Ctrl). b Igrafu yeBar ibonisa ubungakanani bokusebenza kweentsuku ze-12 inkcubeko yeposi kuzo zonke iimeko ze-4 zenkcubeko (Ctrl, TD, MC, kunye ne-MT) xa kuthelekiswa nezilayi zentliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kunye ne-D12 MT), 12 (D12 MC) izilayi / iqela le-0 , # ANOVA, uvavanyo olulodwa 0; ###p <0.001 xa kuthelekiswa ne-D0 kunye ne-**p <0.01 xa kuthelekiswa ne-D12 ctrl). b Гистограмма показывает количественную оценку жизнеспособности через 12 дней после культивирования во всех 4 ю со свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 CTRL, D12 TD kunye D12 MT), 12 (D12 MC) срезов/группу от разных свиней #0тей свиней, программы #дисто#дисто#дисто#VAсты ,0001, ###p < 0,001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). b Igrafu yebha ibonisa ubungakanani bokusebenza kwiintsuku ze-12 inkcubeko yeposi kuzo zonke iimeko zenkcubeko ye-4 (ulawulo, TD, MC, kunye ne-MT) xa kuthelekiswa namacandelo entliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, kunye ne-D12 MT), 12 (D12 MC) ukusuka kwi- # 00 . 1, ###p <0.001 vs. D0 kunye **p <0.01 ngokuthelekisa D12 Ctrl). b 条形图显示所有4 种培养条件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) (n = 18 (D0)、15 (D12 Ctrl MT1,D12 自和2 MC) 切片/组，进行单向ANOVA 测试;b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия культивирования (контроль, TD, MC ne MT) по сравнению со свежими срезами сердца (D0) (n = 18 (D12), CTRL 18 (D12) разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA; b I-Histogram ebonisa zonke iimeko zenkcubeko ye-4 (ulawulo, i-TD, i-MC kunye ne-MT) xa kuthelekiswa namacandelo entliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kunye ne-D12 MT), ukusuka kwiingulube ezahlukeneyo 12 (D12 MC) amacandelo / iqela, uvavanyo olulodwa lwe-ANOVA; # # 0 #0 # 0 D<p. 0.01 vs. ulawulo D12). c Igrafu yeBar ibonisa ubungakanani be-glucose flux yeentsuku ze-12 i-post culture kuzo zonke iimeko ze-4 zenkcubeko (Ctrl, TD, MC, kunye ne-MT) xa kuthelekiswa nezilayi zentliziyo entsha (D0) (n = 6 izilayi / iqela elivela kwiihagu ezahlukeneyo, uvavanyo olulodwa lwe-ANOVA lwenziwa; ###p <0.001, xa kuthelekiswa ne-D0 kunye ne-*** 1 xa kuthelekiswa ne-D0 kunye ne-*** 1 Ctrl 1 xa kuthelekiswa ne-D0 p. c Igrafu yeBar ibonisa ubungakanani be-glucose flux yeentsuku ze-12 i-post culture kuzo zonke iimeko ze-4 zenkcubeko (Ctrl, TD, MC, kunye ne-MT) xa kuthelekiswa nezilayi zentliziyo entsha (D0) (n = 6 izilayi / iqela elivela kwiihagu ezahlukeneyo, uvavanyo olulodwa lwe-ANOVA lwenziwa; ###p <0.001, xa kuthelekiswa ne-D0 kunye ne-*** 1 xa kuthelekiswa ne-D0 kunye ne-*** 1 Ctrl 1 xa kuthelekiswa ne-D0 p. глююк мерез 12 колес кулич кулес кулич кулес кулич ования (контроль, TD, MC и) сра нц (D = 6 групзуй свй свине от сви ыппоолттттттттстсссссссс те; ;тя тетя тетя ano. # # 1 c I-Histogram ibonisa ubungakanani be-glucose flux iintsuku ze-12 emva kwenkcubeko phantsi kwazo zonke iimeko zenkcubeko ye-4 (ulawulo, i-TD, i-MC kunye ne-MT) xa kuthelekiswa namacandelo entliziyo entsha (D0) (n = 6 amacandelo / iqela elivela kwiingulube ezahlukeneyo, uvavanyo olulodwa lwe-ANOVA lwenziwa; c 条形图显示所有4 种培养条件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) 相比，培养条件) 6 片/组，来自不同猪，单向执行ANOVA 测试；###p <0.001，与D0 相比，***p <0.001 与D12 Ctrl 相比）. C 条形图 显示 所有 4 种 条件 （ ctrl 、 td 、 mc 和 mt） 新鲜 心脏 切片 切片 切片 切片 1的 通量 定量 （n = 6 片/组 , 來自 猪 , , , , , , , 猪 猪单行向执ANOVA 测试；###p <0.0001 ,##p <0.0001 Xhuma i-D12 Ctrl 相比）. c Гистограмма, показывающая количественную оценку потока глюкозы через 12 дней после культивирования для всех 4 ю со свежими срезами сердца (D0) (n = 6 срезов/группа, от разных свиней, односторонний Были проведены тесты ANOVA; ###p < 0,001 I-12 (контроль). c I-Histogram ebonisa ubungakanani be-glucose flux kwiintsuku ze-12 emva kwenkcubeko kuzo zonke iimeko zenkcubeko ye-4 (ulawulo, i-TD, i-MC, kunye ne-MT) xa kuthelekiswa namacandelo entliziyo entsha (D0) (n = 6 amacandelo / iqela, ukusuka kwiingulube ezahlukeneyo, i-unilateral Ngaba iimvavanyo ze-ANOVA zenziwa, ###p <0.001 xa kuthelekiswa ne-D0. ***1 xa kuthelekiswa no-D0.d Iziqendu zokuhlalutya koxinzelelo lwentsha (eluhlaza okwesibhakabhaka), usuku lwe-12 MC (luhlaza), kunye nosuku lwe-12 MT (obomvu) lwezicubu kwiindawo ezilishumi zecandelo leethishu zengingqi (n = 4 izilayi/iqela, uvavanyo lwendlela enye ye-ANOVA; kwakungekho mahluko ubalulekileyo phakathi kwamaqela).e Isakhiwo se-Volcano esibonisa izakhi zofuzo ezichazwe ngokwahlukileyo kumacandelo entliziyo entsha (D0) xa kuthelekiswa namacandelo entliziyo akhuliswe phantsi kweemeko ezimileyo (Ctrl) okanye phantsi kweemeko zeMT (MT) iintsuku eziyi-10-12.f Imephu yobushushu yesarcomere yofuzo yamacandelo entliziyo akhuliswe phantsi kweemeko zenkcubeko nganye.Iibha zeemposiso zibonisa intsingiselo ± ukutenxa okusemgangathweni.
Ukuxhomekeka kwe-Metabolic ekutshintsheni ukusuka kwi-fatty acid oxidation ukuya kwi-glycolysis luphawu lwe-cardiomyocyte dedifferentiation.I-cardiomyocytes ekhulileyo isebenzisa i-glucose kwimveliso ye-ATP kwaye ibe ne-hypoplastic mitochondria kunye ne-cristae5,32 embalwa.Uhlalutyo lokusetyenziswa kwe-glucose lubonise ukuba phantsi kweemeko ze-MC kunye ne-MT, ukusetyenziswa kwe-glucose kwakufana naloo mini ye-0 izicubu (Umfanekiso 4c).Nangona kunjalo, iisampulu zeCtrl zibonise ukwanda okukhulu kokusetyenziswa kweglucose xa kuthelekiswa nezicubu ezitsha.Oku kubonisa ukuba ukudibanisa kwe-CTCM kunye ne-T3 / Dex kwandisa ukusebenza kwezicubu kunye nokugcina i-phenotype ye-metabolism ye-12-day cultured heart section.Ukongezelela, uhlalutyo loxinzelelo lubonise ukuba amanqanaba oxinzelelo ahlala efana nezicubu zentliziyo entsha kwiintsuku ze-12 phantsi kweemeko zeMT kunye ne-MS (umzobo 4d).
Ukuhlalutya impembelelo epheleleyo ye-CTCM kunye ne-T3 / Dex kwi-transcriptal landscape yomhlaba wonke ye-cardiac slice tissue, senze i-RNAseq kwi-cardiac slices kuzo zonke iimeko ezine zeenkcubeko ezahlukeneyo (iDatha eyoNgezelelweyo 1).Okubangel 'umdla kukuba, amacandelo e-MT abonise ukufana okuphezulu kokubhaliweyo kwizicubu zentliziyo entsha, kunye ne-16 kuphela echazwe ngokwahlukileyo kwii-13,642 genes.Nangona kunjalo, njengoko sibonisile ngaphambili, i-Ctrl izilayi zibonise i-1229 izakhi zofuzo ezichazwe ngokwahlukileyo emva kweentsuku ze-10-12 kwinkcubeko (Umfanekiso 4e).Ezi datha zaqinisekiswa yi-qRT-PCR yentliziyo kunye ne-fibroblast genes (i-Supplementary Fig. 7a-c).Okubangel 'umdla kukuba, amacandelo e-Ctrl abonise ukuthotywa kwe-cardiac kunye ne-cell cycle genes kunye nokusebenza kweenkqubo zofuzo ezivuthayo.Ezi datha zibonisa ukuba ukuhlukana, okwenzeka ngokuqhelekileyo emva kokucwangciswa kwexesha elide, kugxininiswe ngokupheleleyo phantsi kweemeko zeMT (i-Supplementary Fig. 8a,b).Uphononongo olucokisekileyo lwezakhi zofuzo ze-sarcomere lubonise ukuba kuphela phantsi kweemeko ze-MT iijene ezifakela i-sarcomere (Umfanekiso we-4f) kunye nesiteshi se-ion (i-Supplementary Fig. 9) igcinwe, ikhusela ekucinezeleni phantsi kwe-Ctrl, TD, kunye neemeko ze-MC.Ezi datha zibonisa ukuba kunye nokudibanisa komatshini kunye nokuvuselela i-humoral (T3 / Dex), i-heart slice transcriptome inokuhlala ifana nezilayi zentliziyo entsha emva kweentsuku ze-12 kwinkcubeko.
Ezi ziphumo ze-transcriptional zixhaswa yinyaniso yokuba ingqibelelo yesakhiwo se-cardiomyocytes kumacandelo entliziyo igcinwe kakuhle phantsi kweemeko ze-MT kwiintsuku ze-12, njengoko kuboniswe nge-connexin ecacileyo kunye ne-43 yendawo (umzobo 5a).Ukongezelela, i-fibrosis kumacandelo entliziyo phantsi kweemeko zeMT yancitshiswa kakhulu xa kuthelekiswa ne-Ctrl kwaye ifana namacandelo entliziyo entsha (umzobo 5b).Ezi nkcukacha zibonisa ukuba ukudibanisa ukuvuselela oomatshini kunye nonyango lwe-T3 / Dex lugcina ngokufanelekileyo isakhiwo senhliziyo kumacandelo entliziyo kwinkcubeko.
a Representative immunofluorescence imifanekiso ye-troponin-T (eluhlaza), i-connexin 43 (ebomvu), kunye ne-DAPI (ebhlowu) kumacandelo entliziyo asanda yedwa (D0) okanye akhuliswe ngeentsuku ze-12 kuzo zonke iimeko ezine zenkcubeko yecandelo lentliziyo (i-scale bar = 100 µm).). I-Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0 kunye ne-D12 Ctrl), 5 (D12 TD, D12 MC kunye ne-D12 MT) izilayi / iqela elivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA lwendlela enye lwenziwa; Ctrl). I-Artificial intelligence quantification of the heart tissue ofstructural integrity (n = 7 (D0 kunye ne-D12 Ctrl), 5 (D12 TD, D12 MC kunye ne-D12 MT) izilayi / iqela elivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA lwendlela enye lwenziwa; Ctrl). Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта (n = 7 (D0 kunye D12 Ctrl), 5 (D12 TD, D12 MC/ D12 MT/гур) ых свиней, проведен однофакторный тест ANOVA; Ubungakanani bengqibelelo yesakhiwo sezicubu zentliziyo kusetyenziswa ubukrelekrele bokwenziwa (n = 7 (D0 kunye neD12 Ctrl), 5 (D12 TD, D12 MC kunye neD12 MT) amacandelo/iqela elisuka kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA lwendlela enye; ).对不同猪的心脏组织结构完整性（n = 7（D0 和D12 CTRL）、5（D12 TD、D12 MC 和D12 MT）切片/组组）切片/组行单向ANOVA 测试；#### p <0.0001 与D0 和*p <0.05 相比，或****p <0.0001 与D12 Ctrl 相比）.对 不同 猪 的 心脏 结构 完整性 （n = 7（d0 和 d12 ctrl） （5（d12 td 、 d12 mc 和 d12 mc 和 d12 mc （ d12 mc）化 进行 单向 单向 单向 测试 ; ########### p <0.0001 与D0 和*p <0.05 相比，或****p <0.00012 .Ubungakanani bengqibelelo yesakhiwo sezicubu zentliziyo usebenzisa i-intelligence yokufakelwa kwiihagu ezahlukeneyo (n = 7 (D0 kunye ne-D12 Ctrl), i-5 (D12 TD, i-D12 MC kunye ne-D12 MT) amacandelo / iqela) kunye novavanyo olulodwa lwe-ANOVA;#### p < 0,0001 по сравнению с D0 и *p < 0,05 okanye ****p < 0,0001 по сравнению с D12 Ctrl). #### p <0.0001 xa kuthelekiswa ne-D0 kunye *p <0.05 okanye ****p <0.0001 xa kuthelekiswa ne-D12 Ctrl). b Imifanekiso emeleyo kunye nobungakanani bentliziyo izilayi ezidyojwe ngebala le-trichrome lika-Masson (i-Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, kunye ne-D12 MC), 9 (D12 MT) izilayi/iqela elisuka kwiihagu ezahlukileyo, indlela enye 0 # D0p ithelekiswa 0 # D0p kwaye ithelekisa 0 # D0p kwaye ithelekisa 0 # D0P 0.001, okanye ****p <0.0001 xa kuthelekiswa ne-D12 Ctrl). b Imifanekiso emeleyo kunye nobungakanani bentliziyo izilayi ezidyojwe ngebala le-trichrome lika-Masson (i-Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, kunye ne-D12 MC), 9 (D12 MT) izilayi/iqela elisuka kwiihagu ezahlukileyo, indlela enye 0 # D0p ithelekiswa 0 # D0p kwaye ithelekisa 0 # D0p kwaye ithelekisa 0 # D0P 0.001, okanye ****p <0.0001 xa kuthelekiswa ne-D12 Ctrl). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Массона (масштабная 50D) = D10D (10D) = D10 Ctrl, D12 TD kunye ne-D12 MC), 9 (D12 MT) срезов/группу от разных свиней, выполняется односторонний тест ANOVA; ####p <0,0001 по D0, *** 1 по сравнению с D12 Ctrl). b Imifanekiso emele kunye nobungakanani bamacandelo entliziyo adyojwe ngebala le-trichrome lika-Masson (isikali sebha = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD kunye ne-D12 MC), 9 (D12 MT) amacandelo/iqela elisuka kwiihagu ezahlukeneyo, lenze indlela enye #00 kunye ne-DANOVA #00. 1 okanye ****p <0.0001 vs. D12 Ctrl). b 用Masson 三色染料染色的心脏切片的代表性图像和量化（比例尺= 500 µm）（n = 10（D2（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D）同猪的9 个（D12 MT）切片/组，进行单因素方差分析;比）. b 用 masson 三 色 染料 的心脏 切片 的 代表性 和量化 （比例 尺 尺 尺 = 500 µm） （n d1（n d1（n（n（d（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（（d0（ d 1（ d 1（ d 1 d 1 d d 1和 d12 mc） 來自 不同 的 9 个 d12 mt 切片 切片 切片 切片 切片 切片 切片 切片 切片 切片 切片片 片 切片 切片 切片/组，进行单因素方差分析；####p <0.0001 与D0 相毅 <0.0001 D0 相毅2 Ctrl 相比）. b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромом Массона (масштабная линейка = 200, D1 = CTRL = 500, D1 = 500, D1 12 MC), 9 (D12 MT) срезов от разных свиней / группы, один- способ ANOVA; b Imifanekiso emele kunye nobungakanani bamacandelo entliziyo adyojwe nge-trichrome ka-Masson (isikali sebha = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD kunye ne-D12 MC), 9 (D12 MT) amacandelo asuka kwiihagu/iqela, enye indlela ye-ANOVA; #0.1 ukuya ku-D0. p <0.0001 xa kuthelekiswa ne-D12 Ctrl).Iibha zeemposiso zibonisa intsingiselo ± ukutenxa okusemgangathweni.
Ekugqibeleni, ukukwazi kwe-CTCM ukulinganisa i-hypertrophy ye-cardiac yavavanywa ngokunyuka kwe-cardiac tissue stretch.Kwi-CTCM, uxinzelelo lwegumbi eliphezulu lomoya lwenyuka ukusuka kwi-80 mmHg ukuya kwi-80 mmHg.Ubugcisa.(ukwelula okuqhelekileyo) ukuya kuthi ga kwi-140 mmHg Art.(Umfanekiso 6a).Oku kuhambelana nokunyuka kwe-32% kwi-stretch (umzobo 6b), eyayiboniswe ngaphambili njengepesenti ehambelanayo yokwelula efunekayo kumacandelo entliziyo ukufezekisa ubude be-sarcomere obufana nobo bubonwa kwi-hypertrophy.Ukolula kunye nesantya sezicubu zenhliziyo ngexesha lokuqhawula kunye nokuphumla kwahlala kuhleli ngexesha leentsuku ezintandathu zenkcubeko (Umfanekiso 6c).Izicubu zenhliziyo ezivela kwiimeko ze-MT zixhomekeke kwi-stretch yesiqhelo (MT (Eqhelekileyo)) okanye iimeko zokugqithisa (MT (OS)) iintsuku ezintandathu.Kakade emva kweentsuku ezine kwinkcubeko, i-hypertrophic biomarker NT-ProBNP yayiphakanyiswe kakhulu phakathi kweemeko eziphantsi kweMT (OS) xa kuthelekiswa neemeko zeMT (eziqhelekileyo) (umzobo 7a).Ukongezelela, emva kweentsuku ezintandathu zokulima, ubukhulu beseli kwi-MT (OS) (Umfanekiso we-7b) unyuke kakhulu xa kuthelekiswa namacandelo entliziyo yeMT (eqhelekileyo).Ukongeza, i-NFATC4 i-nuclear translocation yanda kakhulu kwii-tissue ezigqithisiweyo (Umfanekiso 7c).Ezi ziphumo zibonisa uphuhliso oluqhubekayo lwe-pathological remodeling emva kwe-hyperdistension kunye nokuxhasa ingcamango yokuba ifowuni ye-CTCM ingasetyenziselwa njengeqonga lokufunda ukubonakaliswa kwe-cardiac hypertrophy.
Umkhondo omele uxinzelelo lwegumbi lomoya, uxinzelelo lwegumbi lolwelo, kunye nemilinganiselo yokunyakaza kwezicubu ziqinisekisa ukuba uxinzelelo lwegumbi lutshintsha uxinzelelo lwegumbi lolwelo, olubangela ukunyakaza okuhambelanayo kwesilayi sethishu.b Ipesenti emeleyo eyoluliweyo kunye neegophe zesantya sokunwebeka ngokuqhelekileyo ukolulwa (orenji) kunye namacandelo eethishu ezithe kratya (blue).c Igrafu yebha ebonisa ixesha lomjikelo (n = 19 izilayi ngeqela ngalinye, ukusuka kwiihagu ezahlukeneyo), ixesha lokunqanda (n = 18-19 izilayi kwiqela ngalinye, ukusuka kwiihagu ezahlukeneyo), ixesha lokuphumla (n = 19 izilayi ngeqela, ukusuka kwiihagu ezahlukeneyo)), i-amplitude ye-tissue movement (n = 14 izilayi / iqela, ukusuka kwiihagu ezahlukeneyo), i-peak 4 ye-slice ye-pigs, i-relax ye-pigs kunye ne-1 n = 14 (D0), 15 (D6) ) amacandelo / amaqela) avela kwiihagu ezahlukeneyo), uvavanyo lwe-t-tailed yoMfundi olunemisila emibini lubonisa ukuba akukho mahluko abalulekileyo kuyo nayiphi na iparameter, ebonisa ukuba ezi parameters zahlala zihleli ngexesha leentsuku ze-6 zenkcubeko kunye nokugqithisa.Iibha zeemposiso zibonisa intsingiselo ± ukutenxa okusemgangathweni.
iBar graph quantification ye-NT-ProBNP yoxinaniso kwimidiya yenkcubeko ukusuka kwintliziyo yeelayiti ezikhuliswe phantsi kweMT eziqhelekileyo zoluleko (Norm) okanye ukugqithisa (OS) iimeko (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kunye ne-D4 MTOS) izilayi / iqela elivela kwiingulube ezahlukeneyo, iindlela ezimbini ze-ANOVA xa kuthelekiswa ne-ANOVA eqhelekileyo. iBar graph quantification ye-NT-ProBNP yoxinaniso kwimidiya yenkcubeko ukusuka kwizilayi zentliziyo ezikhuliswe phantsi kweMT eziqhelekileyo zoluleko (Norm) okanye ukugqithisa (i-OS) iimeko (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kunye ne-D4 MTOS) izilayi / iqela elivela kwiihagu ezahlukeneyo, i-Two-way-way 1 xa kuthelekiswa ne-ANOVA0;Ubungakanani be-histogram ye-NT-ProBNP yoxinaniso kwinkcubeko yenkcubeko ukusuka kwizilayi zentliziyo ezikhuliswe phantsi kweemeko eziqhelekileyo ze-MT stretch (norm) okanye i-overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kunye ne-D4) .MTOS) izilayi / iqela elivela kwiihagu ezahlukeneyo, uhlalutyo lwezinto ezimbini zenziwa ngokungafaniyo;**p < 0,01 по сравнению с нормальным растяжением). **p <0.01 xa kuthelekiswa nokwelula okuqhelekileyo). a 在MT 正常拉伸(Eqhelekileyo) 或过度拉伸(OS) 条件下培养的心脏切片培养基中NT-ProBNP 浓度的条序的条彈3 （D2 MTOS、D4 MTNorm 和D4 MTOS）來自不同猪的切片/组，进行双向方差分析；**与正常拉伸相比，p. Ubungakanani be-NT-ProBNP yoxinaniso kwizilayi zentliziyo ezikhuliswe phantsi kweMT eqhelekileyo yoluleko (Eqhelekileyo) okanye i-OS) iimeko (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm neD4 MTOS) ukusuka ezahlukeneyo 猪的切片/组，可以双向双双， p <0.01).i-histogram Ubungakanani be-NT-ProBNP yogxininiso kwiinqununu zentliziyo ezikhuliswe phantsi kweemeko ze-MT eziqhelekileyo eziqhelekileyo (eziqhelekileyo) okanye zigqithise (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) kunye ne-D4 MTOS) izilayi / iqela elivela kwiihagu ezahlukeneyo, uhlalutyo lweendlela ezimbini zokuhluka;**p < 0,01 по сравнению с нормальным растяжением). **p <0.01 xa kuthelekiswa nokwelula okuqhelekileyo). b Imifanekiso emele i-heart slices engcoliswe nge-troponin-T kunye ne-WGA (ngasekhohlo) kunye nobungakanani besayizi yeseli (ekunene) (n = 330 (D6 MTOS), i-369 (D6 MTNorm) iiseli / iqela ukusuka kwi-10 iingcezu ezahlukeneyo kwiihagu ezahlukeneyo, uvavanyo lwe-t-tailed yoMfundi emibini luyenziwa xa kuthelekiswa ne-0 ****01. b Imifanekiso emele i-heart slices engcoliswe nge-troponin-T kunye ne-WGA (ngasekhohlo) kunye nobungakanani besayizi yeseli (ekunene) (n = 330 (D6 MTOS), i-369 (D6 MTNorm) iiseli / iqela ukusuka kwi-10 iilayiti ezahlukeneyo kwiihagu ezahlukeneyo, Uvavanyo lwe-t-tailed yoMfundi emibini luyenziwa xa kuthelekiswa ne-0 ****01. b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественного определения размера клеток (6) = D6 (D6) MTNorm) клеток/группу из 10 разных срезов от разных свиней, два- проводится хвостовой t-критерий Стьюдента; ****p <0,00001   b Imifanekiso emeleyo yamacandelo entliziyo afakwe kwi-troponin-T kunye ne-AZP (ngasekhohlo) kunye nobungakanani besayizi yeseli (ekunene) (n = 330 (D6 MTOS), i-369 (i-D6 MTNorm) iiseli / iqela ukusuka kumacandelo e-10 ahlukeneyo kwiihagu ezahlukeneyo, i-t-tailed ye-t-test yoMfundi emibini ye-t-test ye-0 xa kuthelekiswa ne-0. b 用肌钙蛋白-T 和WGA（左）和细胞大小量化（右)个不同切片的369（D6 MTNorm）细胞/组，两进行有尾学生t 检验；与正常拉伸相比，****p <0.00. b Imifanekiso emele izilayi zentliziyo ene-calcarein-T kunye ne-WGA (ekhohlo) kunye nobukhulu beseli (ekunene) (n = 330 (D6 MTOS), i-369 ukusuka kwi-10 yamacandelo ahlukeneyo (i-D6 MTNorm)) Iiseli / 组，两方法有尾学，pa 0 kunye novavanyo oluqhelekileyo ; b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественная оценка размера клеток (639 MT = MT63) rm) из 10 различных срезов от разных свиней Клетки/группа, двусторонние критерий Стьюдента; ****p < 0,0001 по сравнению с номмальные). b Imifanekiso emele yamacandelo entliziyo enziwe nge-troponin-T kunye ne-AZP (ekhohlo) kunye nobungakanani besayizi yeseli (ekunene) (n = 330 (D6 MTOS), 369 (D6 MTNorm) ukusuka kumacandelo e-10 ahlukeneyo kwiihagu ezahlukeneyo) Iiseli / iqela, imilinganiselo enemisila emibini t;****p <0.0001 xa kuthelekiswa noxinzelelo oluqhelekileyo). c Imifanekiso emele usuku lwe-0 kunye nosuku lwe-6 I-MTOS yentliziyo izilayi ze-immunolabeled ye-troponin-T kunye ne-NFATC4 kunye nobungakanani bokudluliselwa kwe-NFATC4 kwi-nuclei ye-CMs (n = 4 (D0), 3 (D6 MTOS) izilayi / iqela elivela kwiihagu ezahlukeneyo, i-two-tailed Student 0. c Imifanekiso emele usuku lwe-0 kunye nosuku lwe-6 I-MTOS yentliziyo izilayi ze-immunolabeled ye-troponin-T kunye ne-NFATC4 kunye nobungakanani be-translocation ye-NFATC4 kwi-nuclei ye-CMs (n = 4 (D0), 3 (D6 MTOS) izilayi / iqela elivela kwiihagu ezahlukeneyo, i-Two-tailed 0 Student. c Репрезентативные изображения для срезов сердца 0 kunye 6 дней MTOS, иммуномеченых для тропонина-Т kunye NFATC4, kunye количественная оценсок зных клеток (n = 4 (D0), 3 (D6 MTOS) срезов/группу от разных свиней , выполняется двусторонний t-критерий Стьюдента, 0 <p. c Imifanekiso emele amacandelo entliziyo kwi-0 kunye neentsuku ze-6 i-MTOS, i-immunolabeled ye-troponin-T kunye ne-NFATC4, kunye ne-quantification ye-NFATC4 yokudluliselwa kwi-nucleus yeeseli ze-cavernous (n = 4 (D0), 3 (D6 MTOS) izilayi / iqela elivela kwiihagu ezahlukeneyo) lenziwe ngabafundi abanemisila emibini;*p <0.05). c 用于肌钙蛋白-T 和NFATC4 免疫标记的第0 天和第6 天MTOS 心脏切片的代表性图像，以及來自背光明的時候的量化（n = 4 (D0)、3 (D6 MTOS) 切片/组, 进行双尾学生t 检验；*p <0.05). c Ummeli wemifanekiso ye-calcanin-T kunye ne-NFATC4 immunolabeling 第0天和第6天MTOS izilayi zentliziyo, kunye ne-NFATC4 evela kwi-NFATC4 eyahlukileyo 易位至CM iseli nucleus的quantity化 (n = 4 (D0), 3 (D6 MTOS) 其兄生et 电影；*p <0.05). c Репрезентативные изображения срезов сердца MTOS ku 0 kunye 6 день для иммуномаркировки тропонином-Т kunye NFATC4 kunye количественная оценская оценка зных свиней (n = 4 (D0), 3 (D6 MTOS) срез/группа, два- хвостатый t-критерий Стьюдента; *p <0,05). c Imifanekiso emele i-MTOS yamacwecwe entliziyo ngosuku lwe-0 kunye ne-6 ye-troponin-T kunye ne-NFATC4 i-immunolabeling kunye ne-quantification ye-NFATC4 yokutshintshwa kwi-nucleus ye-CM ukusuka kwiihagu ezahlukeneyo (n = 4 (D0), 3 (D6 MTOS) izilayi / iqela, i-t-tailed two-tailed t -0p .Iibha zeemposiso zibonisa ± ukutenxa okusemgangathweni.
Uphando lwe-cardiovascular translational lufuna iimodeli zeselula ezivelisa ngokuchanekileyo indawo yenhliziyo.Kule sifundo, isixhobo se-CTCM saphuhliswa kwaye sibonakaliswe esinokuvuselela amacandelo e-ultrathin entliziyo.Inkqubo ye-CTCM ibandakanya i-physiologically synchronized electromechanical stimulation kunye ne-T3 kunye ne-Dex fluid enrichment.Xa amacandelo entliziyo ye-porcine evezwe kwezi zinto, ukusebenza kwazo, ukunyaniseka kwesakhiwo, umsebenzi we-metabolic, kunye nokuchazwa kwe-transcriptional kwahlala kufana nakwizicubu zentliziyo entsha emva kweentsuku ze-12 zenkcubeko.Ukongeza, ukolula okugqithisileyo kwezicubu zentliziyo kunokubangela i-hypertrophy yentliziyo ebangelwa yi-hyperextension.Ngokubanzi, ezi ziphumo zixhasa indima ebalulekileyo yeemeko zenkcubeko ye-physiological ekugcineni i-phenotype ye-cardiac eqhelekileyo kunye nokubonelela ngeqonga lokuhlola iziyobisi.
Izinto ezininzi zinegalelo ekudaleni indawo efanelekileyo yokusebenza kunye nokusinda kwe-cardiomyocytes.Eyona nto ibonakalayo kwezi zinto zihambelana (1) ukusebenzisana kwe-intercellular, (2) ukuvuselela i-electromechanical, (3) izinto ezihlekisayo, kunye (4) ne-metabolic substrates.Unxibelelwano lwePhysiological cell-to-cell lufuna uthungelwano oluntsonkothileyo olunamacala amathathu eentlobo ezininzi zeeseli ezixhaswa yimatrix engaphandle kwecellular.Ukusebenzisana kweselula okunjalo kunzima ukuphinda kwakhiwe kwakhona kwi-vitro ngenkcubeko ye-co-culture yeentlobo zeeseli zomntu ngamnye, kodwa kunokufezekiswa ngokulula ngokusebenzisa i-organotypic nature yamacandelo entliziyo.
Ukolulwa komatshini kunye nokuvuselela umbane we-cardiomyocytes kubaluleke kakhulu ekugcineni i-phenotype yenhliziyo33,34,35.Ngelixa uvuselelo lomatshini sele lusetyenziswa ngokubanzi kwi-hiPSC-CM imeko kunye nokuvuthwa, izifundo ezininzi ezintle kutshanje ziye zazama ukuvuselela izilayi zentliziyo kwinkcubeko zisebenzisa ukulayisha uniaxial.Ezi zifundo zibonisa ukuba i-2D uniaxial mechanical loading inefuthe elihle kwi-phenotype yentliziyo ngexesha lenkcubeko.Kwezi zifundo, amacandelo entliziyo aye alayishwa nge-isometric tensile forces17, i-linear auxotonic loading18, okanye umjikelo we-cardiac waphinda wenziwa kusetyenziswa impendulo ye-force transducer kunye ne-tension drives.Nangona kunjalo, ezi ndlela zisebenzisa i-uniaxial tissue stretch ngaphandle kokulungiswa kwendalo, okubangela ukunyanzeliswa kweejeni ezininzi zentliziyo okanye ukugqithiswa kweemfuza ezihambelana neempendulo ezingaqhelekanga.I-CTCM echazwe apha inikezela nge-3D i-electromechanical stimulus elingisa umjikelezo wendalo we-cardiac ngokwexesha lokujikeleza kunye ne-physiological stretch (25% stretch, 40% systole, 60% diastole, kunye ne-72 beats ngomzuzu).Nangona oku kukhuthazwa komatshini we-three-dimensional yodwa akwanelanga ukugcina ingqibelelo yezicubu, indibaniselwano ye-humoral kunye ne-mechanical stimulation usebenzisa i-T3 / Dex iyadingeka ukuze kugcinwe ngokufanelekileyo ukusebenza kwezicubu, ukusebenza kunye nokunyaniseka.
Izinto ezihlekisayo zidlala indima ebalulekileyo ekumodareyitheni i-phenotype yentliziyo yabantu abadala.Oku kwagxininiswa kwizifundo ze-HiPS-CM apho i-T3 kunye ne-Dex zongezwa kwimidiya yenkcubeko ukukhawulezisa ukukhula kweeseli.I-T3 inokuphembelela ukuthuthwa kwee-amino acids, iswekile kunye ne-calcium kwi-cell membranes36.Ukongezelela, i-T3 ikhuthaza ukubonakaliswa kwe-MHC-α kunye nokunciphisa i-MHC-β, ukukhuthaza ukubunjwa kwe-myofibrils ngokukhawuleza kwi-cardiomyocytes ekhulile xa kuthelekiswa ne-slow twitch myofibrils kwi-CM yomntwana.Ukunqongophala kwe-T3 kwizigulane ze-hypothyroid kubangela ukulahlekelwa kweebhendi ze-myofibrillar kunye nokunciphisa izinga lokuphuhliswa kwethoni37.I-Dex isebenza kwii-receptors ze-glucocorticoid kwaye ibonakaliswe ukwandisa ukunyanzeliswa kwe-myocardial kwiintliziyo ezinqabileyo ezizimeleyo;I-38 le mpucuko icingelwa ukuba ihambelana nefuthe kwi-calcium deposit-driven entry (SOCE) 39,40.Ukongeza, i-Dex ibophelela kwii-receptors zayo, ibangela impendulo ebanzi ye-intracellular ecinezela ukusebenza komzimba kunye nokuvutha30.
Iziphumo zethu zibonisa ukuba ukukhuthazwa komatshini womzimba (MS) kuphuculwe ukusebenza kwenkcubeko jikelele xa kuthelekiswa neCtrl, kodwa ayiphumelelanga ukugcina ukusebenza, ukunyaniseka kwesakhiwo, kunye nokubonakaliswa kwentliziyo kwiintsuku ze-12 kwinkcubeko.Xa kuthelekiswa ne-Ctrl, ukongezwa kwe-T3 kunye ne-Dex kwiinkcubeko ze-CTCM (MT) ziphucule ukusebenza kunye nokugcinwa kweeprofayili zokubhalwa okufanayo, ukunyaniseka kwesakhiwo, kunye nomsebenzi we-metabolic kunye nezicubu zentliziyo entsha kwiintsuku ze-12.Ukongezelela, ngokulawula iqondo le-tissue stretch, i-hyperextension-induced cardiac hypertrophy model yenziwe ngokusebenzisa i-STCM, ebonisa ukuguquguquka kwenkqubo ye-STCM.Kufuneka kuqatshelwe ukuba nangona ukulungiswa kwentliziyo kunye ne-fibrosis ngokuqhelekileyo kubandakanya izitho ezithintekayo ezineeseli ezijikelezayo ezinokubonelela ngee-cytokines ezifanelekileyo kunye ne-phagocytosis kunye nezinye izinto ezihlengahlengiswayo, amacandelo entliziyo angakwazi ukulinganisa inkqubo ye-fibrotic ekuphenduleni uxinzelelo kunye nokuphazamiseka.kwi-myofibroblasts.Oku kuye kwavavanywa ngaphambili kule modeli yesilayi senhliziyo.Kufuneka kuqatshelwe ukuba iiparamitha ze-CTCM zinokuguqulwa ngokutshintsha uxinzelelo / i-amplitude yombane kunye nokuphindaphinda ukulinganisa iimeko ezininzi ezifana ne-tachycardia, i-bradycardia, kunye nenkxaso ye-mechanical circulatory (intliziyo engalayishwanga).Oku kwenza isixokelelwano sibe siphakathi ekuvavanyweni kweziyobisi.Ukukwazi kwe-CTCM ukulinganisa i-hypertrophy ye-cardiac overexertion-induced cardiac ivula indlela yokuvavanya le nkqubo yonyango lomntu.Ukuqukumbela, uphononongo lwangoku lubonisa ukuba ukunwenwa koomatshini kunye nokuvuselela i-humoral kubalulekile ekugcineni inkcubeko yamacandelo ezicubu zentliziyo.
Nangona idatha eboniswe apha ibonisa ukuba i-CTCM liqonga elithembisayo lokubonisa i-myocardium engaguqukiyo, le ndlela yenkcubeko inemida ethile.Umda oyintloko wenkcubeko ye-CTCM kukuba ibeka uxinzelelo oluqhubekayo lomatshini kwizilayi, ezithintela ukukwazi ukubeka iliso ngokunyanisekileyo ukunyanzeliswa kwe-cardiac slice ngexesha lomjikelo ngamnye.Ukongezelela, ngenxa yobukhulu obuncinci bamacandelo enhliziyo (i-7 mm), ukukwazi ukuvavanya umsebenzi we-systolic ngaphandle kweenkqubo zenkcubeko usebenzisa i-sensor yamandla endabuko ilinganiselwe.Kumbhalo-ngqangi wangoku, sisoyisa ngokuyinxenye lo mda ngokuvavanya amandla ombane okukhanya njengesalathisi somsebenzi wekhontrakthi.Nangona kunjalo, lo mda uya kufuna umsebenzi othe kratya kwaye unokuqwalaselwa kwixesha elizayo ngokuzisa iindlela zokubeka iliso kwintsebenzo yezilayi zentliziyo kwinkcubeko, njengemephu ye-optical usebenzisa i-calcium kunye ne-voltage-sensitive dyes.Omnye umda we-CTCM kukuba imodeli yokusebenza ayisebenzisi uxinzelelo lwe-physiological (preload and afterload).Kwi-CTCM, uxinzelelo luye lwanyanzeliswa ngeendlela ezichaseneyo zokuvelisa i-25% ye-physiological stretch kwi-diastole (i-stretch egcweleyo) kunye ne-systole (ubude be-contraction ngexesha lokuvuselela umbane) kwizicubu ezinkulu kakhulu.Lo mda kufuneka ususwe kuyilo lwe-CTCM oluzayo ngoxinzelelo olwaneleyo kwizicubu zenhliziyo ezivela kumacala omabini kunye nokusebenzisa uxhulumaniso oluchanekileyo-umthamo olwenzeka kumagumbi entliziyo.
Uhlengahlengiso olwenziweyo olugqithisiweyo oluxelwe kulo mbhalo wesandla lulinganiselwe ekulinganiseni imiqondiso ye-hypertrophic hyperstretch.Ngaloo ndlela, le modeli inokunceda ekufundeni kwe-stretch-induced hypertrophic signaling ngaphandle kwesidingo sezinto ezihlekisayo okanye ze-neural (ezingekho kule nkqubo).Uphononongo olongezelelweyo lufunekayo ukunyusa ubuninzi be-CTCM, umzekelo, ukudibanisa kunye neeseli zokuzivikela, ukujikeleza i-plasma humoral factor, kunye nokugcinwa kwangaphakathi xa ukudibanisa kunye neeseli ze-neuronal kuya kuphucula amathuba okuba nesifo somzekelo we-CTCM.
Kusetyenziswe iihagu ezilishumi elinesithathu kolu phononongo.Zonke iinkqubo zezilwanyana zenziwa ngokuhambelana nezikhokelo zeziko kwaye zamkelwa yiYunivesithi yaseLouisville Institutional Animal Care and Use Committee.I-aortic arch yayigxininiswe kwaye intliziyo yaxutywa nge-1 L ye-cardioplegia eyinyumba (110 mM NaCl, 1.2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 U / mL heparin, pH ukuya kwi-7.4); iintliziyo zigcinwe kwisisombululo se-cardioplegic ebandayo de zihanjiswe kwilebhu emkhenkceni oqhelekileyo <10 min. iintliziyo zigcinwe kwisisombululo se-cardioplegic ebandayo de zihanjiswe kwilebhu emkhenkceni oqhelekileyo <10 min. сердца хранили в ледяном кардиоплегическом растворе до транспортировки в лабораторию на льду, что обычно занимает <10 мин. iintliziyo zigcinwe kwisisombululo se-cardioplegic ebandayo kuze kube yilapho ukuthutha ukuya kwibhubhoratri kwi-ice, edla ngokuthatha <10 min.将心脏保存在冰冷的心脏停搏液中，直到冰上运送到实验室，通常<10分钟.将心脏保存在冰冷的心脏停搏液中，直到冰上运送到实验室，通常<10分钟. Держите сердца в ледяной кардиоплегии до транспортировки в лабораторию на льду, обычно <10 мин. Gcina iintliziyo kwi-ice cardioplegia de uthuthelwe kwibhubhoratri kwiqhwa, ngokuqhelekileyo <10 min.
Isixhobo se-CTCM saphuhliswa kwi-software ye-SolidWorks encediswa yikhompyutha (CAD).Amagumbi enkcubeko, abahluli kunye namagumbi omoya ayenziwe ngeplastiki ye-acrylic ecacileyo ye-CNC.I-7mm ye-diameter back-up ring yenziwe nge-polyethylene ephezulu yoxinaniso (HDPE) embindini kwaye ine-o-ring groove yokufaka i-silicone o-ring esetyenziselwa ukutywina imidiya ngaphantsi.I-membrane ye-silica encinci ihlukanisa igumbi lenkcubeko kwi-plate yokwahlukana.I-membrane ye-silicone isikwe nge-laser ukusuka kwi-0.02 ″ ishidi elishinyeneyo le-silicone kwaye inobulukhuni be-35A.Umzantsi kunye neegaskets eziphezulu ze-silicone zisikwe nge-laser ukusuka kwi-1/16 ″ ishidi elishinyeneyo le-silicone kwaye linobulukhuni be-50A.I-316L izikrufu zensimbi engenasici kunye namandongomane aphiko zisetyenziselwa ukubopha ibhloko kunye nokudala itywina elingangeni moya.
Ibhodi yesekethe eprintiweyo ezinikeleyo (PCB) yenzelwe ukudibanisa nenkqubo ye-C-PACE-EM.Iziseko zokudibanisa umatshini we-swiss kwi-PCB ziqhagamshelwe kwi-electrode yegraphite ngeengcingo zobhedu ezifakwe ngesilivere kunye nezikrufu ze-0-60 zobhedu ezifakwe kwi-electrode.Ibhodi yesekethe eprintiweyo ibekwe kwikhava yeprinta ye-3D.
Isixhobo se-CTCM silawulwa yi-programmable pneumatic actuator (PPD) eyenza uxinzelelo olujikelezayo olulawulwayo olufana nomjikelezo wenhliziyo.Njengoko uxinzelelo ngaphakathi kwegumbi lomoya luyanda, i-membrane ye-silicone eguquguqukayo iyanda ukuya phezulu, inyanzelisa i-medium phantsi kwendawo ye-tissue.Indawo yezicubu iya kwandiswa ngoku kugxothwa kolwelo, ukulinganisa ukwanda komzimba wentliziyo ngexesha le-diastole.Kwincopho yokuphumla, ukuvuselela kombane kwasetyenziswa nge-graphite electrode, eyanciphisa uxinzelelo kwigumbi lomoya kwaye yabangela ukucutshungulwa kwamacandelo ezicubu.Ngaphakathi kombhobho i-valve ye-hemostatic ene-sensor yoxinzelelo ukuze ibone uxinzelelo kwinkqubo yomoya.Uxinzelelo olubonwa yinzwa yoxinzelelo lusetyenziswa kumqokeleli wedatha oqhagamshelwe kwilaptop.Oku kuvumela ukujonga ngokuqhubekayo uxinzelelo ngaphakathi kwegumbi legesi.Xa uxinzelelo lwegumbi eliphezulu lufikelelwe (umgangatho we-80 mmHg, i-140 mmHg OS), isixhobo sokufumana idatha sayalelwa ukuba sithumele isignali kwinkqubo ye-C-PACE-EM ukuvelisa i-biphasic voltage signal ye-2 ms, ibekwe kwi-4 V.
Amacandelo entliziyo afunyenwe kunye neemeko zenkcubeko kwimigodi ye-6 zenziwe ngolu hlobo lulandelayo: Dlulisa iintliziyo ezivuniweyo ukusuka kwisitya sokudlulisa kwisitya esinebanda (4 ° C.) i-cardioplegia.I-ventricle ekhohlo yayihlulwe kunye ne-blade oyinyumba kwaye yanqunyulwa ibe yizicucu ze-1-2 cm3.Ezi zithintelo zezicubu zifakwe kwi-tissue supports kunye ne-tissue adhesive kwaye zifakwe kwi-vibrating microtome tissue bath equkethe isisombululo seTyrode kunye ne-oksijini eqhubekayo (3 g / L 2,3-butanedione monooxime (BDM), 140 mM NaCl (8.18 g) . ), 6 mM KCl (0.101M g-glucose), 6 mM KCl (0.101M D-glucose), 0. (2.38 g), 1 mM MgCl2 (1 ml 1 M isisombululo), 1.8 mM CaCl2 (1.8 ml 1 M isisombululo), ukuya kwi-1 L ddH2O).I-microtome engcangcazelayo yayimiselwe ukusika ama-300 µm amazilayi angqindilili kumaza e-80 Hz, i-amplitude ye-vibration ethe tyaba eyi-2 mm, kunye nesantya sangaphambili se-0.03 mm/s.Ibhafu yethishu yayijikelezwe ngumkhenkce ukugcina isisombululo sipholile kwaye ubushushu bugcinwe kwi-4 ° C.Dlulisela amacandelo eethishu ukusuka kwibhafu ye-microtome ukuya kwibhafu yokufukamela enesisombululo esiqhubekayo seTyrode eneoksijini kumkhenkce de kufumaneke amacandelo aneleyo epleyiti yenkcubeko enye.Kwiinkcubeko ze-transwell, amacandelo e-tissue afakwe kwi-sterile 6 mm ububanzi be-polyurethane inkxaso kwaye afakwe kwi-6 ml ye-optimized medium (199 medium, 1x ITS supplement, 10% FBS, 5 ng / ml VEGF, 10 ng / ml FGF-alkaline kunye ne-2X i-antibiotic-antifungal).Ukukhuthazwa kombane (i-10 V, i-frequency 1.2 Hz) isetyenziswe kumacandelo e-tissue ngokusebenzisa i-C-Pace.Kwiimeko ze-TD, i-T3 entsha kunye ne-Dex zongezwa kwi-100 nM kunye ne-1 μM kutshintsho oluphakathi ngalunye.I-medium igcwele i-oxygen ngaphambi kokutshintshwa ngamaxesha ama-3 ngosuku.Amacandelo e-tissue akhuliswe kwi-incubator kwi-37 ° C kunye ne-5% CO2.
Kwiinkcubeko ze-CTCM, amacandelo e-tissue abekwe kwiprinta ye-3D eyenziwe ngokwezifiso kwisitya sePetri esinesisombululo seTyrode esilungisiweyo.Isixhobo senzelwe ukwandisa ubungakanani beqhekeza lentliziyo nge-25% yendawo yeringi yenkxaso.Oku kwenziwa ukwenzela ukuba amacandelo entliziyo angoluleli emva kokudluliselwa kwisisombululo seTyrode ukuya kwindawo ephakathi kunye nangexesha le-diastole.Kusetyenziswa iglu ye-histoacrylic, amacandelo angama-300 µm ubukhulu aye aqiniswa kwiringi yenkxaso eyi-7 mm ububanzi.Emva kokuba unamathele amacandelo e-tissue kwindandatho yokuxhasa, nqumla amacandelo e-tissue engaphezulu kwaye ubeke amacandelo e-tissue aqhotyoshelweyo emva kwi-bath ye-Tyrode isisombululo kwi-ice (4 ° C) de kube amacandelo aneleyo alungiselelwe isixhobo esinye.Ixesha elipheleleyo lokucubungula zonke izixhobo akufanele lidlule iiyure ze-2.Emva kokuba iziqendu ze-6 ze-tissue zifakwe kwiindandatho zabo zokuxhasa, isixhobo se-CTCM sihlanganiswe.Igumbi lenkcubeko ye-CTCM lizaliswe kwangaphambili nge-21 ml ephakathi kwe-oxygenated medium.Dlulisa amacandelo e-tissue kwigumbi lenkcubeko kwaye ususe ngokucophelela naziphi na iibhubhu zomoya nge-pipette.Icandelo le-tissue likhokelwa lingene emngxunyeni kwaye licinezelwe ngobumnene kwindawo.Ekugqibeleni, faka i-cap ye-electrode kwisixhobo kwaye uthumele isixhobo kwi-incubator.Emva koko udibanise i-CTCM kwi-tube yomoya kunye nenkqubo ye-C-PACE-EM.I-actuator ye-pneumatic ivula kwaye ivalve yomoya ivula i-CTCM.Inkqubo ye-C-PACE-EM yenzelwe ukuhambisa i-4 V kwi-1.2 Hz ngexesha le-biphasic pacing ye-2 ms.Isixhobo satshintshwa kabini ngosuku kwaye i-electrodes yatshintshwa kanye ngosuku ukuphepha ukuqokelela kwegraphite kwii-electrodes.Ukuba kuyimfuneko, amacandelo eethishu anokususwa kwimithombo yawo yenkcubeko ukukhupha nawaphi na amaqamza omoya asenokuba awe phantsi kwawo.Kwiimeko zonyango lwe-MT, i-T3 / Dex yongezwa ngokutsha kunye noshintsho oluphakathi kunye ne-100 nM T3 kunye ne-1 μM Dex.Izixhobo ze-CTCM zikhuliswe kwi-incubator kwi-37 ° C kunye ne-5% CO2.
Ukufumana iitrajectories ezolulekileyo zentliziyo, kuye kwaphuhliswa inkqubo ekhethekileyo yekhamera.Ikhamera ye-SLR (Canon Rebel T7i, Canon, Tokyo, Japan) isetyenziswe nge-Navitar Zoom 7000 18-108mm macro lens (Navitar, San Francisco, CA).Umbono wenziwa kwiqondo lobushushu begumbi emva kokutshintsha indawo ephakathi nendawo entsha.Ikhamera ibekwe kwi-angle ye-51 ° kwaye ividiyo irekhodwa kwiifreyimu ze-30 ngesekhondi.Okokuqala, isoftware yomthombo ovulekileyo (MUSCLEMOTION43) yasetyenziswa kunye noMfanekiso-J ukulinganisa intshukumo yezilayi zentliziyo.Imaski yenziwa kusetyenziswa iMATLAB (MathWorks, Natick, MA, USA) ukuchaza iindawo ezinomdla zokubetha izilayi zentliziyo ukunqanda ingxolo.Iimaski ezahlulwe ngesandla zisetyenziswa kuyo yonke imifanekiso kulandelelwano lwesakhelo kwaye emva koko igqithiselwe kwiplagi ye-MUSCLEMOTION.I-Muscle Motion isebenzisa i-avareji yobunzima beepikseli kwisakhelo ngasinye ukulinganisa intshukumo yayo ngokunxulumene nesakhelo sereferensi.Idatha yarekhodwa, yahluzwa kwaye isetyenziselwa ukulinganisa ixesha lokujikeleza kunye nokuvavanya i-tissue stretch ngexesha lomjikelezo wenhliziyo.Ividiyo erekhodiweyo yalungiswa emva kokuba kusetyenziswa i-odolo yokuqala ye-zero yesigaba sokucoca idijithali.Ukulinganisa i-tissue stretch (i-peak-to-peak), uhlalutyo lwe-peak-to-peak lwenziwa ukuhlula phakathi kweencopho kunye nemibhobho kwisignali erekhodiweyo.Ukongeza, ukurhoxiswa kwenziwa kusetyenziswa i-6th order polynomial ukuphelisa i-signal drift.Ikhowudi yenkqubo yaphuhliswa kwiMATLAB ukumisela ukunyakaza kwezicubu zehlabathi, ixesha lokujikeleza, ixesha lokuphumla, kunye nexesha lokunciphisa (iKhowudi yeNkqubo eyoNgezelelweyo ye-44).
Uhlalutyo loxinzelelo, usebenzisa iividiyo ezifanayo ezenzelwe ukuvavanya ukunwebeka komatshini, siqale silandele imifanekiso emibini emele iincopho zentshukumo (ezona ndawo ziphakamileyo (eziphezulu) nezisezantsi (ezisezantsi) ngokwesoftware ye-MUSCLEMOTION.Emva koko sahlula imimandla yezicubu kwaye sisebenzise ifom ye-algorithm ye-shading kwi-tissue edibeneyo (i-Supplementary Fig. 2a).I-tissue edibeneyo yahlula-hlulwa ibe yi-subsurfaces ezilishumi, kwaye uxinzelelo kwindawo nganye lubalwe kusetyenziswa i-equation elandelayo: Uxinzelelo = (i-Sup-Sdown) / i-Sdown, apho i-Sup kunye ne-Sdown yimida yemo ukusuka phezulu kunye ne-shadow shadows of the fabric, ngokulandelanayo (i-Supplementary Fig. 2b).
Iziqendu zentliziyo zilungiswe kwi-4% ye-paraformaldehyde kwiiyure ze-48.Izicubu ezisisigxina zaphelelwa ngamanzi kwi-10% kunye ne-20% ye-sucrose kwi-1 h, emva koko kwi-30% ye-sucrose ngobusuku.Amacandelo ke afakwe kwi-optimum cutting temperatures compound (i-OCT compound) kwaye ngokuthe ngcembe iqhwale kwi-isopentane / i-ice ice bath.Gcina iibhloko zokuzinzisa ze-OCT kwi--80 °C de kube kukwahlulwa.Izilayidi zalungiswa njengamacandelo anobunzima be-8 μm.
Ukususa i-OCT kumacandelo entliziyo, fudumeza izilayidi kwibhloko yokufudumeza kwi-95 °C kwi-5 min.Yongeza i-1 ml ye-PBS kwisilayidi ngasinye kwaye ufukamele imizuzu engama-30 kwiqondo lobushushu begumbi, uze ungene kumacandelo ngokuseta i-0.1% ye-Triton-X kwi-PBS imizuzu eyi-15 kwiqondo lobushushu begumbi.Ukuthintela ama-antibodies angabonakaliyo ekubopheni isampuli, yongeza i-1 ml ye-3% yesisombululo se-BSA kwiislayidi kwaye udibanise iyure eli-1 kwindawo yokushisa.I-BSA yasuswa kwaye izilayidi zahlanjwa nge-PBS.Phawula isampuli nganye ngepensile.I-antibodies eziphambili (i-diluted 1: 200 kwi-1% BSA) (connexin 43 (Abcam; #AB11370), i-NFATC4 (Abcam; #AB99431) kunye ne-troponin-T (i-Thermo F Scientific; #MA5-12960) yongezwa ngaphezu kwemizuzu ye-90, emva koko i-4% ye-Alexaluted antibodies kwi-12% ye-mouse ye-mouse: 8 (Thermo Scientific; #A16079), ngokuchasene nomvundla Alexa Fluor 594 (Thermo Scientific; #T6391) imizuzu eyongezelelweyo ye-90 Ihlanjwe amaxesha e-3 nge-PBS Ukwahlula ibala elijoliswe kuyo kwimvelaphi, sasebenzisa kuphela i-antibody yesibini njengolawulo. -x magnification) kunye ne-Keyence microscope ene-40x yokwandisa.
I-WGA-Alexa Fluor 555 (i-Thermo Scientific; #W32464) kwi-5 μg / ml kwi-PBS yayisetyenziselwa i-WGA staining kwaye isetyenziswe kumacandelo asisigxina kwi-30 imizuzu kwiqondo lokushisa.Izilayidi zaye zahlanjwa nge-PBS kwaye iSudan emnyama yongezwa kwisilayidi ngasinye kwaye yafukanyelwa imizuzu engama-30.Izilayidi zaye zahlanjwa nge-PBS kunye ne-vectashield embedding medium yongezwa.Izilayidi zabonwa kwi-microscope ye-Keyence kwi-40x yokukhulisa.
I-OCT isusiwe kwiisampuli njengoko kuchazwe ngasentla.Emva kokususa i-OCT, ntywilisela izilayidi kwisisombululo sikaBouin ngobusuku.Izilayidi zaye zihlanjululwe ngamanzi adibeneyo ngeyure eli-1 kwaye emva koko zifakwe kwi-Bibrich aloe acid fuchsin isisombululo semizuzu eyi-10.Emva koko iislayidi zihlanjwe ngamanzi adibeneyo kwaye zifakwe kwisisombululo se-5% phosphomolybdenum / 5% phosphotungstic acid imizuzu eyi-10.Ngaphandle kokuhlamba, dlulisela izilayidi ngqo kwisisombululo se-aniline eluhlaza okwesibhakabhaka imizuzu eyi-15.Emva koko iislayidi zihlanjwe ngamanzi adibeneyo kwaye zifakwe kwi-1% isisombululo se-acetic acid imizuzu emi-2.Izilayidi zomiswa kwi-200 N ethanol kwaye zigqithiselwe kwi-xylene.Izilayidi ezingcolileyo zabonwa kusetyenziswa i-microscope ye-Keyence kunye nenjongo ye-10x.Ipesenti yendawo yeFibrosis yalinganiswa kusetyenziswa isoftware yeKeyence Analyzer.
I-CyQUANT™ MTT Uvavanyo Lokusebenza Kweseli (Invitrogen, Carlsbad, CA), inombolo yekhathalogu V13154, ngokweprothokholi yomenzi enohlengahlengiso oluthile.Ngokukodwa, i-punch yokuhlinzwa kunye nobubanzi be-6 mm yayisetyenziselwa ukuqinisekisa ubungakanani bezicubu ezifanayo ngexesha lohlalutyo lwe-MTT.Izicubu zaye zafakwa ngabanye kwiqula le-12-well plate ene-MTT substrate ngokweprotocol yomenzi.Amacandelo afakwe kwi-37 ° C. kwiiyure ze-3 kwaye izicubu eziphilayo zixuba i-substrate ye-MTT ukwenza i-purple formazan compound.Faka esikhundleni sesisombululo se-MTT nge-1 ml ye-DMSO kwaye ufukame kwi-37 °C imizuzu eyi-15 ukukhupha i-formazan emfusa kumacandelo entliziyo.Iisampulu zahlanjululwa kwi-1: 10 kwi-DMSO kwiiplate ezisezantsi ezicacileyo ze-96 kunye nokuqina kombala omfusa kulinganiswe kwi-570 nm usebenzisa i-Cytation plate reader (BioTek).Ufundo lwenziwa ngokwesiqhelo kubunzima besilayi ngasinye sentliziyo.
Imidiya yesilayi sentliziyo yatshintshwa ngemithombo yeendaba equlethe i-1 μCi/ml [5-3H] -glucose (Moravek Biochemicals, Brea, CA, USA) yovavanyo lokusetyenziswa kweglucose njengoko kuchaziwe ngaphambili.Emva kweeyure ezi-4 zokufukamela, yongeza i-100 µl ephakathi ukuya kwityhubhu ye-microcentrifuge evulekileyo equlethe i-100 µl ye-0.2 N HCl.Emva koko ityhubhu yafakwa kwityhubhu ye-scintillation equlethe i-500 μl ye-dH2O ukuze ibe ngumphunga [3H] 2O iiyure ezingama-72 kwi-37°C.Emva koko susa ityhubhu ye-microcentrifuge kwi-tube ye-scintillation kwaye ungeze i-10 ml ye-scintillation fluid.Ubalo lwe-scintillation lwenziwa kusetyenziswa i-Tri-Carb 2900TR liquid scintillation analyzer (Inkampani yePackard Bioscience, iMeriden, CT, USA).Ukusetyenziswa kweglucose kuye kwabalwa kuthathelwa ingqalelo [5-3H] -umsebenzi othile weglucose, ukulingana okungaphelelanga kunye nemvelaphi, ukuhlanjululwa kwe- [5-3H] -ukuya kwi-glucose engabhalwanga, kunye nokusebenza kakuhle kwe-scintillation counter.Idatha iqhelekile kubunzima bamacandelo entliziyo.
Emva kwe-tissue homogenization e-Trizol, i-RNA yahlukaniswa kumacandelo entliziyo isebenzisa i-Qiagen miRNeasy Micro Kit #210874 ngokweprotocol yomenzi.Ukulungiswa kwethala leencwadi le-RNAsec, ulandelelwano kunye nohlalutyo lwedatha lwenziwa ngolu hlobo lulandelayo:
I-1 μg ye-RNA ngesampulu isetyenziswe njengesixhobo sokuqala ukulungiselela ithala leencwadi le-RNA.Ulandelelwano lwamathala eencwadi aveliswe kusetyenziswa i-NEBNext UltraTM RNA Library Preparation Kit ye-Illumina (NEB, USA) ngokulandela iingcebiso zomenzi, kunye neekhowudi zesalathisi zongezwa kulandelelwano lweempawu kwisampulu nganye.Ngokufutshane, i-mRNA yahlanjululwa kwi-RNA iyonke kusetyenziswa amaso amagnetic adityaniswe ne-poly-T oligonucleotides.Uqhekeko lwenziwa kusetyenziswa ii-divalent cations kubushushu obuphezulu kwi-NEBNext First Strand Synthesis Reaction Buffer (5X).I-cDNA yokuqala yomtya yadityaniswa kusetyenziswa iiprimer zehexamer ezingahleliwe kunye ne-M-MuLV reverse transcriptase (RNase H-).I-cDNA ye-strand yesibini emva koko idityaniswe kusetyenziswa i-DNA polymerase I kunye ne-RNase H. I-overhangs eseleyo iguqulwa ibe yiziphelo ezibuhlungu nge-exonuclease / polymerase umsebenzi.Emva kwe-adenylation ye-3 'isiphelo se-DNA fragment, i-Adapter ye-NEBNext ene-hairpin loop structure ifakwe kuyo ukuze ilungiselele ukuxutywa.Ukukhethwa kwamaqhekeza e-cDNA ubude obukhethiweyo 150-200 bp.iziqwenga zamathala eencwadi zacocwa kusetyenziswa inkqubo ye-AMPure XP (Beckman Coulter, Beverly, USA).Emva koko, i-3 μl USER Enzyme (NEB, USA) enesayizi-ekhethiweyo ye-cDNA edityaniswe neadaptha yasetyenziswa imizuzu eyi-15 ku-37°C kwaye emva kwemizuzu emi-5 kwi-95°C phambi kwe-PCR.I-PCR iye yenziwa kusetyenziswa i-Phusion High-Fidelity DNA polymerase, iiprimers ze-PCR zendalo yonke, kunye ne-Index (X) primers.Ekugqibeleni, iimveliso ze-PCR zahlanjululwa (inkqubo ye-AMPure XP) kunye nomgangatho wethala leencwadi uhlolwe kwi-Agilent Bioanalyzer 2100 inkqubo.Ilayibrari ye-cDNA emva koko yalandelelwaniswa kusetyenziswa i-Novaseq sequencer.Iifayile zemifanekiso ekrwada ezivela kwi-Illumina zaguqulelwa ekubeni zifundeke ekrwada kusetyenziswa iCASAVA Base Calling.Idatha ekrwada igcinwe kwiifayile zefomathi ye-FASTQ(fq) equlethe ulandelelwano lokufunda kunye neempawu ezisisiseko ezihambelanayo.Khetha i-HISAT2 ukuze utshatise ulandelelwano oluhluziweyo lufundeka kwi-Sscrofa11.1 reference genome.Ngokubanzi, i-HISAT2 ixhasa i-genomes nakweyiphi na isayizi, kubandakanya i-genomes enkulu kune-4 yeebhiliyoni zeziseko, kwaye amaxabiso angagqibekanga amiselwe uninzi lweeparamitha.I-Splicing ifundeka ukusuka kwidatha ye-RNA Seq inokulungelelaniswa ngokufanelekileyo usebenzisa i-HISAT2, inkqubo ekhawulezayo ekhoyo ngoku, ngokuchaneka okufanayo okanye okungcono kunayo nayiphi na enye indlela.
Ubuninzi bemibhalo ebhaliweyo bubonisa ngokuthe ngqo inqanaba lokubonakaliswa kofuzo.Amanqanaba embonakalo yemfuza avavanywa ngobuninzi bemibhalo ebhaliweyo (ubalo lolandelelwano) olunxulunyaniswa negenome okanye i-exons.Inani elifundiweyo lilingana nomgangatho wokuchazwa kofuzo, ubude bemfuza, kunye nobunzulu bolandelelwano.I-FPKM (amaqhekeza ngewaka ngalinye isiseko sezibini ezibhaliweyo ezilandelelaniswe ngokwesigidi sezibini ezisisiseko) zibaliwe kwaye amaxabiso e-P okuchaza umahluko amiselwa kusetyenziswa iphakheji ye-DESeq2.Emva koko sibala izinga lokufumanisa ubuxoki (FDR) kwixabiso ngalinye le-P usebenzisa indlela ye-Benjamin-Hochberg9 ngokusekelwe kwi-R-function eyakhelwe ngaphakathi "p.adjust".
I-RNA eyodwa kumacandelo entliziyo yaguqulwa yaba yi-cDNA ekugxininiseni kwe-200 ng / μl usebenzisa i-SuperScript IV Vilo Master mix from Thermo (Thermo, cat. no. 11756050).Ubungakanani be-RT-PCR benziwa ngokusebenzisa i-Applied Biosystems Endura Plate Microamp 384-well transparent reaction plate (Thermo, cat. No. 4483319) kunye ne-microamp optical adhesive (Thermo, ikati. No. 4311971).Umxube wokusabela uquka i-5 µl Taqman Fast Advanced Master mix (Thermo, ikati # 4444557), 0.5 µl Taqman Primer kunye ne-3.5 µl H2O exutywe ngequla ngalinye.Imijikelezo eqhelekileyo ye-qPCR yaqhutywa kwaye amaxabiso e-CT alinganiswa kusetyenziswa i-Applied Biosystems Quantstudio 5 real-time PCR isixhobo (384-well module; imveliso # A28135).I-Taqman Primers ithengwe kwi-thermo (i-GAPDHH (SS03375), iPkd1038_M1), i-PKDC2), i-PGB1 H), GATA4 (SS03383805), GJA1 (SS03375), i-ARM03375
Ukukhutshwa kwemidiya ye-NT-ProBNP kwavavanywa kusetyenziswa ikiti ye-NT-ProBNP (ingulube) (iCat. No. MBS2086979, MyBioSource) ngokweprotocol yomenzi.Ngokufutshane, i-250 µl yesampulu nganye kunye nomgangatho wongezwa ngokuphindwa kwequla ngalinye.Ngokukhawuleza emva kokongeza isampuli, yongeza i-50 µl ye-Assay Reagent A kwiqula ngalinye.Gcoba ngobumnene ipleyiti kwaye utywine nge-sealant.Emva koko iipilisi zifakwe kwi-37 ° C ngeyure eli-1.Emva koko ufune isisombululo kwaye uhlambe amaqula amaxesha ama-4 nge-350 µl yesisombululo sokuhlamba se-1X, ngokufukamela isisombululo sokuhlamba imizuzu eyi-1-2 ngexesha ngalinye.Emva koko yongeza i-100 µl ye-Assay Reagent B ngequla ngalinye kwaye utywine ngepleyiti yokutywina.Ithebhulethi yashukunyiswa ngobumnene kwaye ifakwe kwi-37 ° C imizuzu engama-30.Funa isisombululo kwaye uhlambe amaqula ka-5 nge-350 µl yesisombululo sokuhlamba se-1X.Yongeza i-90 µl yesisombululo sesubstrate kwiqula ngalinye kwaye uvale ipleyiti.Gcoba iplate kwi-37 ° C ngemizuzu eyi-10-20.Yongeza 50 µl Misa Isisombululo kwiqula ngalinye.Ipleyiti yalinganiswa ngokukhawuleza usebenzisa i-Cytation (BioTek) umfundi weplate ebekwe kwi-450 nm.
Uhlalutyo lwamandla lwenziwa ukukhetha ubungakanani beqela eliya kubonelela> 80% amandla okubona i-10% yotshintsho olupheleleyo kwiparameter kunye ne-5% yohlobo lwempazamo ye-I. Uhlalutyo lwamandla lwenziwa ukukhetha ubungakanani beqela eliya kubonelela> 80% amandla okubona i-10% yotshintsho olupheleleyo kwiparameter kunye ne-5% yohlobo lwempazamo ye-I. Анализ мощности был выполнен для выбора размеров групп, которые обеспечат >80% мощности для обнаружения 10% абеспечат той ошибок типа I. Uhlalutyo lwamandla lwenziwa ukukhetha ubungakanani beqela eliza kubonelela> i-80% amandla okubona i-10% inguqu epheleleyo yeparameter kunye ne-5% yohlobo lwempazamo ye-I.进行功效分析以选择将提供> 80％功效以检测参数中10％绝对变化和5％I型错误率的。进行功效分析以选择将提供> 80％功效以检测参数中10％绝对变化和5％I型错误率的。 Был проведен анализ мощности для выбора размера группы, который обеспечил бы > 80% мощности для обнаружения 10% проведен астоты ошибок типа I. Uhlalutyo lwamandla lwenziwa ukukhetha ubungakanani beqela eliza kubonelela> 80% amandla okubona i-10% yokutshintsha ipharamitha epheleleyo kunye ne-5% yohlobo lwe-I yempazamo yezinga.Amacandelo e-tissue akhethwe ngokungenamkhethe phambi kovavanyo.Lonke uhlalutyo lwaluyimeko eyimfama kwaye iisampulu zaqondwa kuphela emva kokuba yonke idatha ihlalutyiwe.Isoftware yeGraphPad Prism (iSan Diego, CA) yasetyenziselwa ukwenza lonke uhlalutyo lwamanani. Kuzo zonke iinkcukacha-manani, amaxabiso e-p athathwa njengento ebalulekileyo kumaxabiso <0.05. Kuzo zonke iinkcukacha-manani, amaxabiso e-p athathwa njengento ebalulekileyo kumaxabiso <0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Kuzo zonke iinkcukacha-manani, amaxabiso e-p athathwa njengento ebalulekileyo kumaxabiso <0.05.对于所有统计数据，p 在值<0.05 时被认為是显着的.对于所有统计数据，p 在值<0.05 时被认為是显着的. Для всей статистики p-значения считались значимыми при значениях <0,05. Kuzo zonke iinkcukacha-manani, amaxabiso e-p athathwa njengento ebalulekileyo kumaxabiso <0.05.Uvavanyo lwe-t loMfundi olunemisila emibini lwenziwa kwidatha ngokuthelekisa oku-2 kuphela.Indlela enye okanye indlela ezimbini i-ANOVA yayisetyenziselwa ukumisela ukubaluleka phakathi kwamaqela amaninzi.Xa usenza iimvavanyo ze-post hoc, ukulungiswa kukaTukey kusetyenziswe kwi-akhawunti yokuthelekisa ezininzi.Idatha ye-RNAsec inengqwalasela ekhethekileyo yamanani xa ubala i-FDR kunye ne-p.adjust njengoko kuchazwe kwicandelo leNdlela.
Ukufumana ulwazi oluthe kratya malunga noyilo lwesifundo, bona iNgxelo yoPhando lweNdalo eqhagamshelwe kweli nqaku.