Urakoze gusura Kamere.com.Verisiyo ya mushakisha ukoresha ifite inkunga ya CSS igarukira.Kuburambe bwiza, turagusaba ko ukoresha mushakisha ivuguruye (cyangwa ugahagarika uburyo bwo guhuza uburyo muri Internet Explorer).Hagati aho, kugirango tumenye gukomeza gushyigikirwa, tuzatanga urubuga rudafite imiterere na JavaScript.
Hano harakenewe sisitemu yizewe ya vitro ishobora kubyara neza ibidukikije byumubiri byumutima kugirango bipimishe ibiyobyabwenge.Kuboneka kwimikorere yumuco wumutima wumuntu byatumye habaho gusobanura nabi ingaruka zibiyobyabwenge byumutima.Hano, twateje imbere icyitegererezo cyumuco wumutima (CTCM) utera amashanyarazi uburyo bwo gukata ibice byumutima kandi bigakorwa muburyo bwa physiologique mugice cya systolique na diastolique yicyiciro cyumutima.Nyuma yiminsi 12 yumuco, ubu buryo bwateje imbere igice cyimitima ibice byumutima, ariko ntibyarinze neza ubusugire bwimiterere.Kubwibyo, nyuma yo gusuzuma molekile ntoya, twasanze kongeramo 100 nM triiodothyronine (T3) na 1 μM dexamethasone (Dex) murwego rwacu rwagumanye microstructure yibice muminsi 12.Hamwe nubuvuzi bwa T3 / Dex, sisitemu ya CTCM yagumanye imyirondoro yinyandiko mvugo, viability, ibikorwa bya metabolike, hamwe nuburinganire bwimiterere kurwego rumwe nuduce twumutima mushya muminsi 12.Byongeye kandi, kurambura gukabije kwimitsi yumutima mumico bitera hypertrophique yumutima yerekana ibimenyetso, bitanga ibimenyetso byubushobozi bwa CTCM bwo kwigana imiterere ya hypertrophique iterwa no kurambura umutima.Mu gusoza, CTCM irashobora kwerekana imiterere ya physiologiya na pathophysiologie yumutima mumuco mugihe kirekire, bigatuma ibizamini byizewe byizewe.
Mbere yubushakashatsi bwubuvuzi, hakenewe kwizerwa muri sisitemu ya vitro ishobora kubyara neza ibidukikije byimiterere yumutima wumuntu.Sisitemu nkiyi igomba kwigana imiterere ihindagurika, umuvuduko wumutima, hamwe na electrophysiologique.Ubwoko bwinyamanswa zikunze gukoreshwa nkurubuga rwo gusuzuma physiologie yumutima hamwe nubwizerwe buke mukugaragaza ingaruka zibiyobyabwenge mumutima wumuntu1,2.Ubwanyuma, Ideal Cardiac Tissue Culture Experimental Model (CTCM) nicyitegererezo cyunvikana cyane kandi cyihariye kubikorwa bitandukanye byo kuvura no kuvura imiti, byerekana neza physiologiya na patrophysiologiya yumutima wumuntu3.Kubura sisitemu nkiyi bigabanya kuvumbura uburyo bushya bwo kunanirwa k'umutima4,5 kandi byatumye imiti yumutima itera nkimpamvu ikomeye yo kuva ku isoko6.
Mu myaka icumi ishize, imiti umunani itari iy'umutima-mitsi yakuwe mu mikoreshereze y’ubuvuzi kuko itera QT intera ndende iganisha kuri arththmias ventricular na rupfu rutunguranye7.Niyo mpamvu, hakenewe ingamba zifatika zo kwipimisha mbere yo gusuzuma imikorere yumutima nimiyoboro.Ikoreshwa rya vuba ryatewe na pluripotent stem selile ikomoka kuri cardiomyocytes (hiPS-CM) mugupima ibiyobyabwenge no gupima uburozi bitanga igisubizo cyikibazo kuri iki kibazo.Nyamara, imiterere idakuze ya hiPS-CMs no kutagira imiterere myinshi yingirangingo yumutima ni imbogamizi zikomeye zubu buryo.Ubushakashatsi buherutse gukorwa bwerekanye ko iyi mbogamizi ishobora kuneshwa hakoreshejwe hiPS-CM hakiri kare gukora hydrogène yumutima yumutima nyuma gato yo gutangira kwikuramo kwizana kandi bigenda byiyongera buhoro buhoro amashanyarazi mugihe runaka.Nyamara, iyi microtissues ya hiPS-CM ibura electrophysiologique ikuze kandi yanduye ya myocardium ikuze.Byongeye kandi, umutima wumutima wumuntu ufite imiterere igoye cyane, igizwe nuruvange rutandukanye rwubwoko butandukanye, harimo selile endothelia selile, neuron, na fibroblast ya tromal, bihujwe nibice bimwe na bimwe bya poroteyine zidasanzwe.Ubu butandukane bwabaturage badafite umutima-11,12,13 mumutima winyamabere ukuze ni inzitizi ikomeye yo kwerekana ingirangingo z'umutima ukoresheje ubwoko bwa selile.Izi mbogamizi zikomeye zishimangira akamaro ko guteza imbere uburyo bwo gutsimbataza ingirabuzimafatizo ya myocardial mu bihe bya physiologique na patologi.
Ibice bito (300 µm) byumutima wumuntu byagaragaye ko ari icyitegererezo cyiza cya myocardium yumuntu.Ubu buryo butanga uburyo bwuzuye bwa sisitemu ya 3D yuzuye imeze nkimitsi yumutima wabantu.Ariko, kugeza muri 2019, gukoresha ibice byumutima byimico byagarutsweho numuco muto (24 h) kubaho.Ibi biterwa nibintu byinshi birimo kubura kurambura kumubiri, gukanika ikirere, no gukoresha itangazamakuru ryoroheje ridashyigikira ibikenerwa byumutima.Muri 2019, amatsinda menshi yubushakashatsi yerekanye ko kwinjiza ibintu byimikorere muri sisitemu yumuco yumutima bishobora kwagura ubuzima bwumuco, kunoza imvugo yumutima, no kwigana indwara yumutima.Ubushakashatsi bubiri bwiza 17 na 18 bwerekana ko gupakira imashini zidasanzwe bigira ingaruka nziza kumutima wa fenotipi mugihe cyumuco.Nyamara, ubu bushakashatsi ntabwo bwakoresheje imbaraga-eshatu-zipima umubiri-wikigereranyo cyumutima wumutima, kubera ko ibice byumutima byashyizwemo imbaraga za isometric tensile 17 cyangwa umurongo wa auxotonic uremereye 18.Ubu buryo bwo kurambura ingirabuzimafatizo bwatumye habaho guhagarika ingirabuzimafatizo nyinshi z'umutima cyangwa gukabya gukabije kwa gen bifitanye isano no kwaduka bidasanzwe.Ikigaragara ni uko Pitoulis n'abandi.19 yateje imbere umutima ucagaguye umuco wo kwiyuhagiriramo kwiyubaka k'umutima ukoresheje ibitekerezo bya transducer ibitekerezo hamwe na drives ya tension.Nubwo iyi sisitemu itanga ibisobanuro birambuye muburyo bwa vitro cardiac cycle yerekana, ibintu bigoye kandi byinjira muburyo bugabanya ikoreshwa rya sisitemu.Laboratoire yacu iherutse gushyiraho uburyo bworoheje bw’umuco hakoreshejwe uburyo bwo gukurura amashanyarazi hamwe n’uburyo bwiza bwo gukomeza ubuzima bw’ibice bya porcine nu mutima wabantu kugeza ku minsi 620,21.
Muri iki gihe cyandikishijwe intoki, turasobanura icyitegererezo cyumuco wumutima (CTCM) dukoresheje ibice byumutima wa porcine ushiramo ibimenyetso byurwenya kugirango wongere usubiremo imiterere yimitima itatu yumutima hamwe no gutandukana kwa patrophysiologique mugihe cyumutima.Iyi CTCM irashobora kongera ubunyangamugayo bwo guhanura ibiyobyabwenge mbere yubuvuzi kurwego rutigeze rugerwaho mugutanga sisitemu yumutima ihenze, hagati yumutima-mutima wigana physiologie / pathophysiologie yumutima winyamabere kugirango bapimwe mbere yubuvuzi.
Ibimenyetso bya Hemodynamic bigira uruhare runini mugukomeza imikorere ya cardiomyocyte muri vitro 22,23,24.Muri iki gihe cyandikishijwe intoki, twateje imbere CTCM (Igishusho 1a) ishobora kwigana ibidukikije byumutima bikuze bitera imbaraga zamashanyarazi nubukanishi kuri radiyo yumubiri (1.2 Hz, gukubitwa 72 kumunota).Kugira ngo wirinde kurambura imyenda ikabije mu gihe cya diastole, hakoreshejwe ibikoresho bya 3D byandika kugira ngo ubunini bwa tissue bugere kuri 25% (Ishusho 1b).Umuvuduko w'amashanyarazi uterwa na sisitemu ya C-PACE wagenewe gutangira ms 100 mbere ya systole ukoresheje sisitemu yo gukusanya amakuru kugirango ubyare byuzuye umutima.Sisitemu yumuco wa tissue ikoresha progaramu ya pneumatike ikora (LB Engineering, Ubudage) kugirango yongere yizenguruke ya silicone yoroheje kugirango itume kwaguka kwimitima yumutima mucyumba cyo hejuru.Sisitemu yahujwe n'umurongo wo mu kirere wo hanze ukoresheje transducer, ituma bishoboka guhindura neza umuvuduko (± 1 mmHg) nigihe (ms 1 ms) (Ishusho 1c).
a Ongeraho igice cya tissue kumpeta ya mm 7 yerekana, yerekanwe mubururu, imbere mucyumba cyumuco cyibikoresho.Icyumba cyumuco gitandukanijwe nicyumba cyikirere na silicone yoroheje.Shira igipapuro hagati ya buri cyumba kugirango wirinde kumeneka.Umupfundikizo wigikoresho urimo electrode ya grafite itanga amashanyarazi.b Igishushanyo cyerekana igikoresho kinini cya tissue, kuyobora impeta nimpeta.Ibice by'imyenda (ibara ry'umukara) bishyirwa ku gikoresho kinini hamwe n'impeta yo kuyobora ishyirwa mu gikoni ku nkombe y'inyuma y'igikoresho.Ukoresheje umurongo ngenderwaho, shyira witonze impeta yingoboka isize hamwe na tissue acrylic yometse hejuru yicyiciro cyumutima.c Igishushanyo cyerekana igihe cyo gukurura amashanyarazi nkigikorwa cyumuvuduko wicyumba cyumuyaga ugenzurwa na programuable pneumatic actuator (PPD).Igikoresho cyo gukusanya amakuru cyakoreshejwe muguhuza ingufu z'amashanyarazi ukoresheje ibyuma byerekana ingufu.Iyo igitutu mucyumba cyumuco kigeze ku mbibi zashyizweho, ikimenyetso cya pulse cyoherejwe kuri C-PACE-EM kugirango gitere amashanyarazi.d Ishusho ya CTCM enye zashyizwe kumurongo wa incubator.Ibikoresho bine bihujwe na PPD imwe binyuze mumuzunguruko wa pneumatike, kandi ibyuma byumuvuduko byinjizwa mumashanyarazi ya hemostatike kugirango bikurikirane umuvuduko wumuzunguruko.Buri gikoresho kirimo ibice bitandatu.
Twifashishije icyuma kimwe cya pneumatike, twashoboye kugenzura ibikoresho 4 bya CTCM, kimwekimwe cyose cyashoboraga gufata ibice 6 (ishusho 1d).Muri CTCM, umuvuduko wumwuka mubyumba byumuyaga uhindurwamo umuvuduko wa syncronique mucyumba cyamazi kandi bigatera kwaguka kwimiterere yumutima (Igice cya 2a na Filime yinyongera 1).Isuzuma ryimyenda irambuye kuri mm 80 Hg.Ubuhanzi.yerekanye kurambura ibice bya tissue kuri 25% (Ishusho 2b).Ijanisha rirambuye ryerekanwe ko rihuye na physiologique sarcomere yuburebure bwa 2.2-22.3 µm kubisanzwe byumutima byumutima 17,19,25.Imyitozo yimyenda yasuzumwe hifashishijwe igenamiterere rya kamera (Ishusho yinyongera).Amplitude n'umuvuduko wo kugenda kwinyama (Ishusho 2c, d) byagereranijwe no kurambura mugihe cyumutima wumutima nigihe mugihe cya systole na diastole (Ishusho 2b).Kurambura n'umuvuduko w'inyama z'umutima mugihe cyo kwikuramo no kuruhuka byagumye bihoraho iminsi 12 mumuco (Ishusho 2f).Kugirango dusuzume ingaruka ziterwa no gukurura amashanyarazi kumasezerano mugihe cyumuco, twashyizeho uburyo bwo kumenya ubumuga bugaragara dukoresheje algorithm igicucu (Igishushanyo cya 2a, b) kandi twashoboye gutandukanya ubumuga hamwe no gukurura amashanyarazi.Igice kimwe cy'umutima (Ishusho 2f).Mu karere cyimukanwa kaciwe (R6-9), voltage mugihe cyo gukurura amashanyarazi yari hejuru ya 20% ugereranije no kubura amashanyarazi, ibyo bikaba byerekana uruhare rwo gukurura amashanyarazi mumikorere.
Ibimenyetso byerekana umuvuduko wicyumba cyumuyaga, umuvuduko wicyumba cyamazi, hamwe nugupima kwimyenda ya tissue byemeza ko umuvuduko wicyumba uhindura umuvuduko wicyumba cyamazi, bigatera kugenda guhuza ibice byumubiri.b Ibimenyetso byerekana ijanisha rirambuye (ubururu) ibice bya tissue bihuye no kurambura ijanisha (orange).c Igipimo cyapimwe cyumutima wumutima gihuye numuvuduko wapimwe wo kugenda.(d) Inzira zerekana inzira ya cycle (umurongo w'ubururu) n'umuvuduko (umurongo utudomo twa orange) mubice byumutima.e Umubare wigihe cyinzira (n = ibice 19 kuri buri tsinda, uhereye ku ngurube zitandukanye), igihe cyo kugabanuka (n = ibice 19 kuri buri tsinda), igihe cyo kuruhuka (n = ibice 19 kuri buri tsinda, uhereye ku ngurube zitandukanye), kugenda kwinyama (n = 25).ibice) / itsinda riva mu ngurube zitandukanye), umuvuduko wa systolike wihuta (n = 24 (D0), 25 (D12) uduce / itsinda riva mu ngurube zitandukanye) nigipimo cyo kuruhuka hejuru (n = 24 (D0), 25 (D12) ibice / itsinda ryingurube zitandukanye).T-ikizamini cyumurizo ibiri yerekanaga nta tandukaniro rinini mubintu byose.f Isesengura ryerekana isesengura ryibice bya tissue hamwe (umutuku) kandi udafite (ubururu) gukurura amashanyarazi, uduce icumi two mukarere twigice cya tissue kuva mugice kimwe.Umwanya wo hasi werekana ingano yijanisha ryijanisha ryingutu mubice bya tissue hamwe no kudatera amashanyarazi mubice icumi uhereye mubice bitandukanye. . . . . （N = 8 片 / 组 ， 来自 不同 的 猪 进行 进行 尾 学生 t 检验 ； **** p <0.0001 ， ** p <0.01 ， * p <0.05）。 （N = 8 片 / 组 ， 来自 不同 的 猪 进行 进行 尾 学生 t 检验 ； **** p <0.0001 ， ** p <0.01 ， * p <0.05）。 . .Utubari twibeshya twerekana uburyo ± gutandukana bisanzwe.
Muri gahunda yacu ya mbere ya biomimetike yumutima uciyemo umuco [20, 21], twakomeje kubaho, imikorere, nuburinganire bwimiterere yimitima yumutima muminsi 6 dukoresheje imbaraga zamashanyarazi no guhuza ibice bito.Ariko, nyuma yiminsi 10, iyi mibare yagabanutse cyane.Tuzerekeza ku bice byimico muri sisitemu yumuco ya biomimetike yabanjirije 20, 21 yo kugenzura (Ctrl) kandi tuzakoresha uburyo bwambere twatezimbere nkibihe bya MC numuco mugihe kimwe cyo gukanika imashini n'amashanyarazi icyarimwe (CTCM).yahamagaye.Ubwa mbere, twahisemo ko gukanika imashini idafite ingufu z'amashanyarazi bidahagije kugirango ubuzima bwimikorere bwiminsi 6 (Ishusho yinyongera 3a, b).Igishimishije, hamwe nogutangiza imbaraga za physio-mashini na mashanyarazi ukoresheje STCM, ubuzima bwibice byumutima byiminsi 12 byakomeje kuba nkibice byumutima mushya mubihe bya MS, ariko ntabwo biri mubihe bya Ctrl, nkuko bigaragazwa nisesengura rya MTT (Ishusho 1).3a).Ibi birerekana ko gukanika imashini hamwe no kwigana inzinguzingo z'umutima bishobora gutuma ibice bigize ingirabuzimafatizo bikomeza kubaho inshuro ebyiri nkuko byavuzwe muri sisitemu yumuco ihamye.Nyamara, gusuzuma uburinganire bwimiterere yibice byinyama hakoreshejwe immunolabeling yumutima troponine T na connexine 43 byagaragaje ko imvugo ya connexine 43 yari hejuru cyane mubice bya MC kumunsi wa 12 kuruta kugenzura kumunsi umwe.Nyamara, imvugo imwe ya connexine 43 hamwe na Z-disikuru ntibyakomeje neza (Ishusho 3b).Twifashishije urwego rwubwenge (AI) kugirango tumenye uburinganire bwimiterere26, umuyoboro ushingiye kumashusho wimbitse ushingiye kuri troponin-T na connexin wanditseho 43 kugirango uhite ugereranya uburinganire bwimiterere na fluorescence yibice byumutima ukurikije imbaraga zaho.Ubu buryo bukoresha imiyoboro ya Neural Network (CNN) hamwe nuburyo bwimbitse bwo kwiga kugirango ugereranye neza uburinganire bwimiterere yimitsi yumutima muburyo bwikora kandi butabogamye, nkuko byasobanuwe mubisobanuro.26. MC tissue yerekanye uburyo bwiza bwo guhuza imiterere kumunsi 0 ugereranije nibice bigenzura.Byongeye kandi, ibara rya trichrome ya Masson ryagaragaje ijanisha rito cyane rya fibrosis mugihe cya MS ugereranije nuburyo bwo kugenzura kumunsi wa 12 wumuco (Ishusho 3c).Mugihe CTCM yongereye imbaraga mubice byumutima byumutima kumunsi wa 12 ikagera kurwego rusa nubwa tissue nshya yumutima, ntabwo byahinduye cyane ubusugire bwimiterere yibice byumutima.
igishushanyo mbonera cyerekana ingano ya MTT yubuzima bushya bwimitima mishya (D0) cyangwa gukata umutima kumico muminsi 12 haba mumico ihamye (D12 Ctrl) cyangwa muri CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), ibice 12 (D12 MC) ibice / ingero ziva kuri D # D1; l). igishushanyo mbonera cyerekana ingano ya MTT yubuzima bushya bwimitima mishya (D0) cyangwa gukata umutima kumico muminsi 12 haba mumico ihamye (D12 Ctrl) cyangwa muri CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), ibice 12 (D12 MC) ibice / ingero ziva kuri D # D1; l).histogramu yerekana ingano yubuzima bwa MTT ibice byumutima mushya (D0) cyangwa umuco wibice byumutima muminsi 12 haba mumico ihamye (kugenzura D12) cyangwa CTCM (D12 MC) (n = 18 (D0), 15 (D12 igenzura).)), ibice 12 (D12 MC) ibice / itsinda biva mu ngurube zitandukanye, hakorwa ikizamini kimwe ANOVA;#### p <0,0001 по сравнению с D0 и ** p <0,01 по сравнению с D12 Ctrl). #### p <0.0001 ugereranije na D0 na ** p <0.01 ugereranije na 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) 中 新鲜 心脏 切片 (D0) 或 心脏 切片 培养 12 天 的 MTT 活力 的 量化 ， OV ((( ， #### p <0.0001 ， 与 D12 Ctrl 相比 ， ** p <0.01)。 a 条形图 显示 在 静态 培养 (D12 Ctrl) 或 CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl) 中 新鲜 心脏 切片 (D0) ， 来自 不同 猪 的 12 (D12 MC) 切片 / 组 ， 进行 单向 OV OV ， ** p。)histogramu yerekana ingano yubuzima bwa MTT mubice bishya byumutima (D0) cyangwa ibice byumutima byatsinzwe muminsi 12 mumico ihamye (D12 igenzura) cyangwa CTCM (D12 MC) (n = 18 (D0), 15 (D12 igenzura)), ibice 12 (D12 MC) ibice / itsinda ryingurube zitandukanye, ikizamini kimwe cya ANOVA;#### p <0,0001 по сравнению с D0, ** p <0,01 по сравнению с D12 Ctrl). #### p <0.0001 ugereranije na D0, ** p <0.01 ugereranije na D12 Ctrl).b Troponin-T (icyatsi), connexine 43 (umutuku) na DAPI (ubururu) mubice byumutima byitaruye (D0) cyangwa ibice byumutima byimico mubihe bihamye (Ctrl) cyangwa CTCM (MC) muminsi 12) byerekana amashusho ya immunofluorescence (igipimo cyuzuye = 100 µm). Ubwenge bwubukorikori bugereranya uburinganire bwimitsi yumutima (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) uduce / itsinda buriwese kuva ingurube zitandukanye, ikizamini kimwe cya ANOVA kirakorwa; #### p <0.0001 ugereranije na D0 na **** p <0.0001 ugereranije na D12 Ctrl). Ubwenge bwubwenge bugereranya uburinganire bwimiterere yumutima (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) uduce / itsinda buri umwe mu ngurube zitandukanye, hakorwa ikizamini kimwe cya ANOVA; #### p <0.0001 ugereranije na D0 na **** p <0.0001 ugereranije na D12 Ctrl). Количественная оценка структурной тоностистисти сердечно китуп п от разых нариней, Проводится одно китыйнтый; ### Umubare wuburinganire bwimiterere yumubiri wumutima ukoresheje ubwenge bwubuhanga (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) ibice / amatsinda yingurube zitandukanye, ikizamini kimwe cya ANOVA cyakozwe; #### p <0.0001 na D0 na **** p <0.0001 ugereranije na D12 Ctrl).人工智能 量化 心脏 组织 结构 完整性 （n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) ibice / itsinda buri ngurube zitandukanye, ikizamini kimwe cya ANOVA; #### p <0.0001 与 D0 相比 ， **** p <0.0001 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) uduce / itsinda buri ngurube zitandukanye, ikizamini kimwe cya ANOVA; #### p <0.0001 与 D0 相比 ， **** p <0.0001 与 D12 Ctrl 相比）。。 Искусственный интеллект для количественной оценки башной целостности сердечной ткани (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) срезов / груу kaждой из разных свиней, одностороны <0,0001 по сравнению с D12 Ctrl). Ubwenge bwa gihanga bwo kugereranya uburinganire bwimiterere yumubiri wumutima (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) ibice / itsinda buri ngurube zitandukanye, ikizamini kimwe cya ANOVA; #### p <0.0001 vs .D0 Kugereranya **** p <0.0001 ugereranije na D12 Ctrl). c Amashusho ahagarariye (ibumoso) no kugereranya (iburyo) kubice byumutima byandujwe na trichrome ya Masson (Scale yambaye ubusa = 500 µm) (n = ibice 10 / itsinda buri umwe mu ngurube zitandukanye, hakorwa ikizamini kimwe cya ANOVA; #### p <0.0001 ugereranije na D0 na *** p <0.001 ugereranije na D12 Ctrl). c Amashusho ahagarariye (ibumoso) no kugereranya (iburyo) kubice byumutima byandujwe na trichrome ya Masson (Scale yambaye ubusa = 500 µm) (n = ibice 10 / itsinda buri umwe mu ngurube zitandukanye, ikizamini kimwe cya ANOVA kirakorwa; #### p <0.0001 ugereranije na D0 na *** p <0.001 ugereranije na D12 Ctrl). c Репрезентныные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красителем Маснаа (чин покрытия = 500 мкм) (n = 10 срезов / кну вот виск) ### p <0,0001 по сравнению с D0 и *** p <0,001 по сравнению с D12 Ctrl). c Amashusho ahagarariye (ibumoso) hamwe no kugereranya (iburyo) yibice byumutima byanditswemo na trichrome ya Masson (igipimo kidafunze = 500 µm) (n = ibice 10 / itsinda riva mu ngurube zitandukanye, ikizamini kimwe cya ANOVA cyakozwe; #### p <0 .0001 ugereranije na D0 na *** p <0.001 ugereranije na D12 Ctrl). c 用 Masson 三 色 染料 染色 的 心脏 切片 代表性 图像 （左） （500 = 500 µm） （n = 10 个 切片 / 组 ， 每组 来自 的 的 猪 ， 进行 单向 ANOVA 。。 C 用 masson 三 色 染料 的 心脏 切片 的 （左 左） 尺度 500 500 500 500 500 = 500 µm） （n = 10 个 切片 组 组 来自 来自 猪 o o o o trl 相比）。 c Репрезентныные изображения (слева) и количественный мат (справа) срезов сердца, окрашенных трихромным красителем Мас медиа (чя шкала = 500 мкм) (n = 10 срезов / группа, кжжот от согг с تالного мата; ### #p <0,0001 по сравнению с D0, *** p <0,001 по сравнению с D12 Ctrl). c Amashusho ahagarariye (ibumoso) numubare (iburyo) wibice byumutima byanditswemo na trichrome ya Masson (ubusa = 500 µm) (n = ibice 10 / itsinda, buri kimwe kiva mu ngurube zitandukanye, cyageragejwe no gusesengura inzira imwe itandukanye; ### # p <0.0001 ugereranije na D0, *** p <0.001 ugereranije na D12 Ctrl).Utubari twibeshya twerekana uburyo ± gutandukana bisanzwe.
Twakekereje ko mugushyiramo molekile ntoya mumico, umuco wa cardiomyocyte ushobora kunozwa kandi iterambere rya fibrosis rikagabanuka mugihe cyumuco wa CTCM.Twasuzumye rero molekile nto dukoresheje imico yacu yo kugenzura static 20,21 kubera umubare muto wibintu bitera urujijo.Dexamethasone (Dex), triiodothyronine (T3), na SB431542 (SB) byatoranijwe kuri iyi ecran.Izi molekile ntoya zagiye zikoreshwa mumico ya hiPSC-CM kugirango itere gukura kwa cardiomyocytes mukongera uburebure bwa sarcomere, T-tubules, n'umuvuduko w'umuvuduko.Byongeye kandi, Dex (glucocorticoid) na SB bizwiho guhagarika umuriro 29,30.Kubwibyo, twagerageje niba gushyiramo imwe cyangwa guhuza izo molekile nto byazamura uburinganire bwimiterere yibice byumutima.Kugirango isuzume ryambere, igipimo cya buri kigo cyatoranijwe hashingiwe ku kwibanda ku buryo bukoreshwa mu ngero z'umuco w'akagari (1 μM Dex27, 100 nM T327, na 2.5 μM SB31).Nyuma yiminsi 12 yumuco, guhuza T3 na Dex byaviriyemo ubunyangamugayo bwiza bwa cardiomyocyte hamwe no kuvugurura fibrous ntoya (Ishusho yinyongera 4 na 5).Byongeye kandi, gukoresha inshuro ebyiri cyangwa ebyiri ibyo bitekerezo bya T3 na Dex byatanze ingaruka zidasanzwe ugereranije nibisanzwe (Ishusho yinyongera. 6a, b).
Nyuma yo kwipimisha kwambere, twakoze igereranya kumutwe kumiterere 4 yimiterere yumuco (Igicapo 4a): Ctrl: ibice byumutima byimico mumico yacu yasobanuwe mbere dukoresheje uburyo bwacu bwiza;20.21 TD: T3 na Ctrl s Yongeyeho Dex kuwa gatatu;MC: ibice byumutima byatsinzwe muri CTCM ukoresheje uburyo bwambere twatezimbere;na MT: CTCM hamwe na T3 na Dex byongewe hagati.Nyuma yiminsi 12 yo guhinga, ubuzima bwimyenda ya MS na MT bwakomeje kuba nkubwa tissue nshya zasuzumwe na MTT (Ishusho 4b).Igishimishije, kwiyongera kwa T3 na Dex mumico ya transwell (TD) ntabwo byatumye habaho iterambere ryinshi mubuzima ugereranije na Ctrl, byerekana uruhare rukomeye rwo gukanika imashini mugukomeza ubuzima bwimitima yumutima.
Igishushanyo mbonera cyerekana igishushanyo mbonera cyerekana imico ine ikoreshwa mugusuzuma ingaruka ziterwa no gukanika imashini hamwe na T3 / Dex byiyongera mugihe cyiminsi 12. b Igishushanyo mbonera cyerekana umubare wibikorwa byiminsi 12 nyuma yumuco nyuma yumuco wose uko ari 4 (Ctrl, TD, MC, na MT) ugereranije nuduce duto twumutima (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD na D12 MT), 12 (D12 MC) uduce / itsinda kuva kuri # 0 # 0 # 0 kuri D12 Ctrl). b Igishushanyo mbonera cyerekana umubare wibikorwa byiminsi 12 nyuma yumuco nyuma yumuco wose uko ari 4 (Ctrl, TD, MC, na MT) ugereranije nuduce duto twumutima (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD na D12 MT), 12 (D12 MC) uduce / itsinda kuva kuri # 0 # 0 # 0 kuri D12 ctrl). b Гистограмма показывает количественную оценку жизнеспособности через 12 дней после митингирования вохех 4 условиях привирирования (дар, TD, MC na MT) по сравнению со свежим срезами срджа с (с) MT), 12. b Igishushanyo mbonera cyerekana ingano yubuzima bwiza muminsi 12 yoherejwe mumico yose uko ari 4 (kugenzura, TD, MC, na MT) ugereranije nibice bishya byumutima (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, na D12 MT), 12 (D12 MC) ibice / itsinda kuva ingurube zitandukanye na # **00 # # 0 rl). 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 、 D12 TD 和 D12 MT) ， 来自 不同 猪 的 12 (D12 MC) 切片0.001 与 D0 相比 ， ** p <0.01 与 D12 控制）。b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия маттивирования (баш, TD, MC na MT) по сравнению со свежим срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD and D12 MT), от ср ср. OVA; #### p <0,0001, ### p <0,001 по сравнению с D0, ** p <0 b Histogramu yerekana imico 4 yose (kugenzura, TD, MC na MT) ugereranije nibice bishya byumutima (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD na D12 MT), uhereye ku ngurube zitandukanye ibice 12 (D12 MC) ibice / itsinda, ikizamini kimwe cya ANOVA; #### p <0.0001, ### p <0.001 vs 0.01. c Igishushanyo mbonera cyerekana ingano ya glucose flux iminsi 12 yoherejwe mumico yose uko ari 4 (Ctrl, TD, MC, na MT) ugereranije nuduce duto twumutima (D0) (n = ibice 6 / itsinda biva mu ngurube zitandukanye, ikizamini kimwe cya ANOVA kirakorwa; ### p <0.001, ugereranije na D0 na *** p <0.001 ugereranije na D12 Ctrl). c Igishushanyo mbonera cyerekana ingano ya glucose flux iminsi 12 yoherejwe mumico yose uko ari 4 (Ctrl, TD, MC, na MT) ugereranije nuduce duto twumutima (D0) (n = ibice 6 / itsinda biva mu ngurube zitandukanye, ikizamini kimwe cya ANOVA kirakorwa; ### p <0.001, ugereranije na D0 na *** p <0.001 ugereranije na D12 Ctrl). c Гистограмма показывает количественную оценку потока глюкозы через 12 дней после митингировано вох 4 условиях медиировирования (дар, TD, MC и MT) по сравнению со свежим срезами срдов оронний Выполняется Тос ANOVA; ### p <0,001 по сравнению с D0 и *** p <0,001 по сравнению с D12 Ctrl). c Histogramu yerekana umubare wa glucose flux iminsi 12 nyuma yumuco nyuma yumuco wose uko ari 4 (kugenzura, TD, MC na MT) ugereranije nibice bishya byumutima (D0) (n = ibice 6 / itsinda biva mu ngurube zitandukanye, ikizamini kimwe cya ANOVA cyakozwe; ### p <0.001 ugereranije na D0 na *** p <0.001 ugereranije na D12 Ctrl). 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 后 天 = n = 6 片 / 组 ， 来自 不同 猪 ， 单向 执行 OV OV l 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 相比 ， 培养 后 后 = n = 6 片 / 组 ， 来自 猪 执行 0.00 0.00 0.00 0.00 0 相比 ， *** p <0.001 与 D12 Ctrl 相比）。 c Гистограмма, показывающая количественную оценку потока глюкозы через 12 дней после митивированя для всех 4 условий митирированя (Amat, TD, MC и MT) по сравнению со свеж с с ср с , односторонний Были проведены Тыы ANOVA; ### p <0,001 по сравнению с D0, *** p <0,001 по сравнению с D12 (бар). c Histogramu yerekana umubare wa glucose flux muminsi 12 nyuma yumuco nyuma yumuco wose uko ari 4 (kugenzura, TD, MC, na MT) ugereranije nibice bishya byumutima (D0) (n = ibice 6 / itsinda, uhereye ku ngurube zitandukanye, Hakozwe ibizamini bya ANOVA muburyo bumwe, ### p <0.001 ugereranije na D0, *** p <0.001 ugereranije na D12 (kugenzura).d Imyanya yo gusesengura ibibanza bishya (ubururu), umunsi wa 12 MC (icyatsi), numunsi wa 12 MT (umutuku) uturemangingo twibice icumi byo mu karere (n = ibice 4 / itsinda, ikizamini kimwe cya ANOVA; nta tandukaniro rikomeye ryari hagati yitsinda).Umugambi wibirunga werekana genes zitandukanye mubice byumutima mushya (D0) ugereranije nibice byumutima byatewe mubihe bihamye (Ctrl) cyangwa mubihe bya MT (MT) muminsi 10-12.f Ubushyuhe bwa genes ya sarcomere kubice byumutima byatewe na buri muco.Utubari twibeshya twerekana uburyo ± gutandukana bisanzwe.
Metabolic iterwa no guhinduranya kuva aside irike ya okiside ikagera kuri glycolysis ni ikiranga umutima wa cardiomyocyte.Cardiomyocytes idakuze ikoresha cyane cyane glucose kubyara ATP kandi ifite mitochondriya ya hypoplastique hamwe na cristae5,32.Isesengura ry'imikoreshereze ya glucose ryerekanye ko mu bihe bya MC na MT, gukoresha glucose byari bisa n'iby'umunsi 0 (Ishusho 4c).Nyamara, ingero za Ctrl zerekanye ubwiyongere bukomeye mu gukoresha glucose ugereranije nuduce dushya.Ibi byerekana ko guhuza CTCM na T3 / Dex byongera imbaraga za tissue kandi bikarinda fenotipike ya metabolike yibice byumutima byimico yiminsi 12.Byongeye kandi, isesengura ry’imiterere ryerekanaga ko urwego rwimitima rwagumye kumera nkurwo mumyanya mishya yumutima muminsi 12 mubihe bya MT na MS (Ishusho 4d).
Kugirango dusesengure ingaruka rusange za CTCM na T3 / Dex kumiterere yisi yose yimiterere yimitsi yumutima, twakoze RNAseq kumice yumutima duhereye kumico ine yose itandukanye (Data Supplementary Data 1).Igishimishije, ibice bya MT byerekanaga ko byandikirwa cyane bisa nuduce twumutima mushya, hamwe 16 gusa bitandukanye muri gen 13,642.Ariko, nkuko twabigaragaje mbere, ibice bya Ctrl byerekanaga genes 1229 zitandukanye nyuma yiminsi 10-12 mumuco (Ishusho 4e).Aya makuru yemejwe na qRT-PCR yumutima na fibroblast (Ishusho yinyongera. 7a-c).Igishimishije, ibice bya Ctrl byerekanaga igabanywa ryimitsi yumutima na selile hamwe no gukora progaramu ya gene inflammatory.Aya makuru yerekana ko gutandukana, ubusanzwe bibaho nyuma yumuco wigihe kirekire, byahujwe rwose mubihe bya MT (Umugereka winyongera 8a, b).Ubushakashatsi bwitondewe bwerekeranye na gen sarcomere bwerekanye ko mubihe bya MT gusa aribwo gen zigizwe na sarcomere (Igishusho 4f) hamwe numuyoboro wa ion (Umugereka wa 9) wabitswe, ubarinda gukandamizwa mubihe bya Ctrl, TD, na MC.Aya makuru yerekana ko hamwe no gukangurira gukanika no gusetsa (T3 / Dex), transcriptice yumutima ishobora gukomeza kumera nkibice byumutima mushya nyuma yiminsi 12 mumuco.
Ibyavuye mu nyandiko-mvugo bishyigikirwa n’uko uburinganire bwimiterere ya cardiomyocytes mu bice byumutima bibikwa neza mugihe cya MT muminsi 12, nkuko bigaragazwa na connexine idahwitse kandi iherereye 43 (Ishusho 5a).Byongeye kandi, fibrosis mubice byumutima mubihe bya MT yagabanutse cyane ugereranije na Ctrl kandi bisa nibice bishya byumutima (Ishusho 5b).Aya makuru yerekana ko guhuza imbaraga zo gukanika no kuvura T3 / Dex birinda neza imiterere yumutima mubice byumutima mumico.
uhagarariye immunofluorescence ishusho ya troponin-T (icyatsi), connexine 43 (umutuku), na DAPI (ubururu) mubice byumutima byitaruye (D0) cyangwa umuco muminsi 12 mubice bine byimitima yumutima (umunzani bar = 100 µm).). Ubwenge bwubwenge bugereranya uburinganire bwimiterere yumutima (n = 7 (D0 na D12 Ctrl), 5 (D12 TD, D12 MC na D12 MT) uduce / itsinda ryingurube zitandukanye, ikizamini kimwe cya ANOVA kirakorwa; #### p <0.0001 ugereranije na D0 na * p <0.05, cyangwa **** p <0.0001 ugereranije na D12 Ctrl. Ubwenge bwubwenge bugereranya uburinganire bwimiterere yumutima (n = 7 (D0 na D12 Ctrl), 5 (D12 TD, D12 MC na D12 MT) uduce / itsinda ryingurube zitandukanye, ikizamini kimwe cya ANOVA kirakorwa; #### p <0.0001 ugereranije na D0 na * p <0.05, cyangwa **** p <0.0001 ugereranije na D12 Ctrl. Количественная оценка структурной церостиги ташености D12 MT) срезов / Группу оп киных кинен, провенты С D12 CTRL). Umubare wuburinganire bwimiterere yumubiri wumutima ukoresheje ubwenge bwubukorikori (n = 7 (D0 na D12 Ctrl), 5 (D12 TD, D12 MC na D12 MT) ibice / itsinda ryingurube zitandukanye, ikizamini kimwe cya ANOVA cyakozwe; #### p <0.0001 ugereranije na D0 na * p <0.05 cyangwa **** p <0.0001 ugereranije na D12 Ctrl).12 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 mt） 组） 人工 智能 量 # # # # # # # # # Ctrl 相比）。Umubare wuburinganire bwimiterere yimitsi yumutima ukoresheje ubwenge bwubukorikori mu ngurube zitandukanye (n = 7 (D0 na D12 Ctrl), 5 (D12 TD, D12 MC na D12 MT) ibice / itsinda) hamwe nikizamini kimwe cya ANOVA;#### p <0,0001 по сравнению с D0 и * p <0,05 или **** p <0,0001 по сравнению с D12 Ctrl). #### p <0.0001 ugereranije na D0 na * p <0.05 cyangwa **** p <0.0001 ugereranije na D12 Ctrl). b uhagarariye amashusho hamwe numubare wumutima wandujwe na Trichrome ya Trichrome ya Trichrome (umuyoboro = 500 (D12 TD) na *** p <0.000 12 ctrl). b uhagarariye amashusho hamwe numubare wumutima wandujwe na Trichrome ya Trichrome ya Trichrome (umuyoboro = 500 (D12 TD) na *** p <0.000 12 ctrl). b Репрезентныные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Мас ина (чнаяяейей = 500 мкм) (n = 10 (D0, D12 Ctrl, D12 TD and D12 MC) я односторонний ту ANOVA; #### p <0,0001 по сравнению с D0 и *** p <0,001 или **** p <0,0001 по сравнению с D12 Ctrl). b Amashusho ahagarariye no kugereranya ibice byumutima byanditseho trichrome ya Masson (umunzani bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD na D12 MC), ibice 9 (D12 MT) ibice / itsinda ryingurube zitandukanye, bakoze inzira imwe ANOVA; #### p <0.0001 na D0 na *** p <0.001 cyangwa ** p. b 用 Masson 三 色 染料 染色 的 心脏 切片 代表性 代表性 图像 和 量化 （= 500 µm） （n = 10 （D0 、 D12 Ctrl 、 D12 TD 和 D12 MC） ， 不同 猪 的 9 个 （ ， *** p <0.001 ， 或 **** p <0.0001 与 D12 Ctrl 相比）。 b 用 masson 三 色 染料 的 心脏 切片 的 和 量化 （= 500 µm） （n = 10 （d0 、 d12 ctrl 、 d12 td 和 d12 mc） 来自 不同 的 个 12 12 12 12 12组 ， 进行 单 因素 方差 分析 ； #### p <0.0001 与 D0 相比 ， *** p <0.001 ， 或 **** p <0.0001 与 D12 Ctrl 相比）。 b Репрезентныные изображения и количественная оценка срезов сердца, окрашенных трихромом Мас хаа (ннаяяейей = 500 мкм) (n = 10 (D0, D12 Ctrl, D12 TD и D12 MC) ср сов ## p <0,0001 по сравнению с D0, *** p <0,001 или **** p <0,0001 по сравнению с D12 Ctrl). b Amashusho ahagarariye no kugereranya ibice byumutima byanditseho trichrome ya Masson (umunzani bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD na D12 MC), ibice 9 (D12 MT) biva mu ngurube / amatsinda atandukanye, uburyo bumwe bwa ANOVA; #### p <0.0001 ugereranije na D0, *** p <0.001 cyangwa **** p <0.001 cyangwa **** p.Utubari twibeshya twerekana uburyo ± gutandukana bisanzwe.
Hanyuma, ubushobozi bwa CTCM bwo kwigana hypertrophyi yumutima byasuzumwe no kongera ingirangingo z'umutima.Muri CTCM, igitutu cyo mu kirere cyo hejuru cyiyongereye kiva kuri 80 mmHg kigera kuri 80 mmHg.Ubuhanzi.(kurambura bisanzwe) kugeza kuri 140 mmHg Ubuhanzi.(Isanamu 6a).Ibi bihuye no kwiyongera kwa 32% kurambura (Igishusho 6b), cyerekanwe mbere nkijanisha rihuye risabwa kugirango ibice byumutima bigere kuburebure bwa sarcomere busa nububoneka muri hypertrophy.Kurambura n'umuvuduko w'inyama z'umutima mugihe cyo kwikuramo no kuruhuka byagumye bihoraho muminsi itandatu yumuco (Ishusho 6c).Imitsi yumutima ituruka kumiterere ya MT yakorewe kurambura bisanzwe (MT (Bisanzwe)) cyangwa kurenza urugero (MT (OS)) muminsi itandatu.Nyuma yiminsi ine mumico, hyparkropique biomarker NT-ProBNP yazamutse cyane muburyo bwa MT (OS) ugereranije na MT (bisanzwe) (Ishusho 7a).Mubyongeyeho, nyuma yiminsi itandatu yumuco, ubunini bwakagari muri MT (OS) (Igishusho 7b) bwiyongereye cyane ugereranije nibice byumutima wa MT (bisanzwe).Byongeye kandi, NFATC4 yo guhindura ibisasu bya kirimbuzi yariyongereye cyane mu myenda irambuye (Ishusho 7c).Ibisubizo byerekana iterambere ryiterambere rya pathologiya nyuma ya hyperdistension kandi bigashyigikira igitekerezo cyuko igikoresho cya CTCM gishobora gukoreshwa nkurubuga rwo kwiga kurambura umutima-hypertrophy yerekana ibimenyetso.
Ibimenyetso byerekana umuvuduko wicyumba cyumuyaga, umuvuduko wicyumba cyamazi, hamwe nugupima kwimyenda ya tissue byemeza ko umuvuduko wicyumba uhindura umuvuduko wicyumba cyamazi, bigatera kugenda guhuza ibice byumubiri.b Uhagarariye kurambura ijanisha no kurambura igipimo cyimirongo isanzwe irambuye (orange) hamwe nuduce twinshi (ubururu).c Igishushanyo mbonera cyerekana igihe cyizunguruka (n = ibice 19 kuri buri tsinda, uhereye ku ngurube zitandukanye), igihe cyo kwikuramo (n = ibice 18-19 kuri buri tsinda, uhereye ku ngurube zitandukanye), igihe cyo kuruhuka (n = ibice 19 kuri buri tsinda, uhereye ku ngurube zitandukanye)), amplitude yimikorere ya tissue (n = 14 ibice / itsinda, biturutse ku ngurube 15 D) ) ibice / amatsinda) biva mu ngurube zitandukanye), t-ikizamini cyumurizo wumunyeshuri yerekanye itandukaniro rinini mubintu byose, byerekana ko ibyo bipimo byagumye bihoraho muminsi 6 yumuco hamwe na volvoltage.Utubari twibeshya twerekana uburyo ± gutandukana bisanzwe.
igipimo cyerekana igishushanyo mbonera cya NT-ProBNP mubitangazamakuru byumuco uhereye kumitima yumutima utewe na MT isanzwe irambuye (Norm) cyangwa kurenza urugero (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, na D4 MTOS) uduce / itsinda riva muburyo butandukanye, ** p <0.01; igipimo cyerekana igishushanyo mbonera cya NT-ProBNP mubitangazamakuru byumuco uhereye kumitima yumutima utewe na MT isanzwe irambuye (Norm) cyangwa kurenza urugero (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, na D4 MTOS) uduce / itsinda riva muburyo butandukanye, ** p <0.01;Umubare wa histogramu ya NT-ProBNP yibanze mumico iciriritse uhereye kumitima yumutima utera imbere mubihe bisanzwe bya MT kurambura (bisanzwe) cyangwa kurenza urugero (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, na D4) .MTOS) ibice / itsinda ryingurube zitandukanye, isesengura ryibintu bibiri bitandukanye;** p <0 ** p <0.01 ugereranije no kurambura bisanzwe). a 在 MT 正常 拉伸 (Norm) 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 条形图 量化 （n = 4 (D2 MTNorm) 、 3 （D2 MTOS 、 D4 MTNorm 和 4 4 p <0.01）。 Quantification ya NT-ProBNP yibanda kumitima yumutima iterwa na MT isanzwe irambuye (Norm) cyangwa kurenza urugero (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm 和 D4 MTOS) bitandukanye na 猪 的 切片 / 组 ， 可以 双向 方 方 发 发 ** **Histogram Quantification yibitekerezo bya NT-ProBNP mubice byumutima byimico mubihe byuburambe busanzwe bwa MT (bisanzwe) cyangwa kurenza urugero (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) na D4 MTOS) ibice / itsinda ryingurube zitandukanye, isesengura ryibice bibiri bitandukanye;** p <0 ** p <0.01 ugereranije no kurambura bisanzwe). b Amashusho agereranya ibice byumutima byanditseho troponin-T na WGA (ibumoso) hamwe nubunini bwa selile (iburyo) (n = 330 (D6 MTOS), 369 (D6 MTNorm) selile / groupe kuva ibice 10 bitandukanye byingurube zitandukanye, ikizamini cya T-umurizo ibiri cyakozwe; **** p <0.0001 ugereranije no kurambura bisanzwe). b Amashusho ahagarariye ibice byumutima byanditseho troponine-T na WGA (ibumoso) hamwe nubunini bwakagari (iburyo) (n = 330 (D6 MTOS), selile 369 (D6 MTNorm) selile / itsinda kuva mubice 10 bitandukanye byingurube zitandukanye, ikizamini cya t-taille ebyiri cyabanyeshuri kirakorwa; **** p <0.0001 ugereranije no kurambura bisanzwe). b Репрезент Канные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественного определения размера клеток (справа) (n = 330 (D6 MTOS), рот рот р водится хвостовой t-критерий Стьюдента; **** p b Amashusho ahagarariye ibice byumutima byanditseho troponin-T na AZP (ibumoso) hamwe nubunini bwakagari (iburyo) (n = 330 (D6 MTOS), selile 369 (D6 MTNorm) selile / itsinda kuva mubice 10 bitandukanye byingurube zitandukanye, ikizamini cya t-umurizo ibiri cyakozwe; **** p <0.0001 ugereranije nuburemere busanzwe). b 用 肌钙蛋白 -T 和 WGA （左） 和 细胞 大小 （右） 染色 = = n = 330 （D6 MTOS） ， 来自 不同 猪 的 个 9 6 6 ** p <0.0001）。 b Amashusho yerekana ibice byumutima byanditseho calcarein-T na WGA (ibumoso) nubunini bwakagari (iburyo) (n = 330 (D6 MTOS), 369 uhereye kumirongo 10 itandukanye (D6 MTNorm)) Ingirabuzimafatizo / 组 ， 两 方法 有 尾 test t ikizamini ； ugereranije no kurambura bisanzwe ， **** p <0.0001). b Репрезент Канные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественная оценка размера клеток (справа) (n = 330 (D6 MTOS), 369 (D6 MTNorm) из 10 ски скичич? niе критерий Стьюдента; **** p <0,0001 по сравнению с କାନныны раст нением). b Amashusho agereranya ibice byumutima byanditseho troponin-T na AZP (ibumoso) no kugereranya ingano ya selile (iburyo) (n = 330 (D6 MTOS), 369 (D6 MTNorm) kuva mubice 10 bitandukanye byingurube zitandukanye) Utugari / itsinda, ibipimo bibiri umurizo t;**** p <0.0001 ugereranije nuburyo busanzwe). c Amashusho ahagarariye kumunsi wa 0 numunsi wa 6 MTOS ikata umutima ikingira immunolabel kuri troponin-T na NFATC4 no kugereranya guhinduranya NFATC4 kuri nuclei ya CMs (n = 4 (D0), 3 (D6 MTOS) uduce / ingurube zitandukanye, t-ikizamini cyabanyeshuri babiri; c Amashusho ahagarariye kumunsi wa 0 numunsi wa 6 MTOS ikata umutima ikingira immunolabel kuri troponin-T na NFATC4 no kugereranya guhinduranya NFATC4 kuri nuclei ya CMs (n = 4 (D0), 3 (D6 MTOS) uduce / ingurube zitandukanye, t-ikizamini cyabanyeshuri babiri; c Репрезентныные изображения для срезов сердца 0 и 6 дней MTOS, im матомеченых для тропонина-Т и NFATC4, и количественная оценка кинлоккации NFATC4 в ядра kaвернозных клеток (n = с) выполняется дву Кононний t-критерий Стьюдента; * p <0,05). c Amashusho ahagarariye ibice byumutima muminsi 0 na 6 MTOS, immunolabel ya troponin-T na NFATC4, hamwe no kugereranya NFATC4 muri nucleus ya selile yubuvumo (n = 4 (D0), 3 (D6 MTOS) uduce / itsinda ryingurube zitandukanye) bakoze ikizamini cya t-umurizo wabanyeshuri babiri;* 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 来自 猪 的 AT NFATC4 易位 至 CM 细胞核 的 量化 （n = 4 (D0) 、 3 D 0.05 0.05 进行 c Amashusho ahagarariye ya calcanin-T na NFATC4 immunolabeling 第 0 天和 第 6 天 MTOS ikata umutima, na NFATC4 kuva NFATC4 易位 至 CM selile nucleus 的 ingano 化 (n = 4 (D0), 3 (D6 MTOS) 切 礼 / 组, 时间 双 尾 学生 学生 学生 c Репрезентныные изображения срезов сердца MTOS на 0 и 6 день для им матомарировки тропонином-Т и NFATC4 и количественная оценка чуклокации NFATC4 в ядра CM от разных свиней (n = 4) ерий Стьюдента; * p <0,05). c Amashusho yerekana amashusho yumutima wa MTOS kumunsi 0 na 6 kuri troponin-T na NFATC4 immunolabeling hamwe no kugereranya kwa NFATC4 muri nucleus ya CM kuva ingurube zitandukanye (n = 4 (D0), 3 (D6 MTOS) uduce / itsinda, umurizo t -criterion Umunyeshuri; * p <0.05);Utubari twibeshya twerekana ± gutandukana bisanzwe.
Ubushakashatsi bwumutima nimiyoboro yimikorere isaba ingirabuzimafatizo zigaragaza neza ibidukikije byumutima.Muri ubu bushakashatsi, hateguwe igikoresho cya CTCM kandi kirangwa gishobora gukangura ultrathin ibice byumutima.Sisitemu ya CTCM ikubiyemo imbaraga za physiologique syncronised electromechanical stimulation hamwe na T3 hamwe na Dex ikungahaye.Iyo ibice byumutima byumutima byagaragaye kuri ibyo bintu, imbaraga zabyo, ubunyangamugayo bwimiterere, ibikorwa bya metabolike, hamwe nimvugo yandikirana byakomeje kuba nkibiri mumitima mishya nyuma yiminsi 12 yumuco.Byongeye kandi, kurambura gukabije kwimitsi yumutima birashobora gutera hypertrophyie yumutima iterwa na hyperextension.Muri rusange, ibisubizo bishyigikira uruhare rukomeye rwimiterere yumuco wa physiologiya mugukomeza fenotipi yumutima isanzwe no gutanga urubuga rwo gusuzuma ibiyobyabwenge.
Ibintu byinshi bigira uruhare mukurema ibidukikije byiza kumikorere no kubaho kwa cardiomyocytes.Ikigaragara cyane muribi bintu bifitanye isano (1) imikoranire hagati yimitsi, (2) gukurura amashanyarazi, (3) ibintu bisetsa, na (4) insimburangingo.Imikorere ya physiologique selile-selile isaba urusobekerane rwibice bitatu-byimiterere yubwoko butandukanye bwunganirwa na matrix idasanzwe.Imikoranire nkiyi igoye iragoye kwiyubaka muri vitro hamwe numuco wubwoko butandukanye, ariko birashobora kugerwaho byoroshye ukoresheje imiterere ya organotypic yibice byumutima.
Kurambura imashini no gukurura amashanyarazi ya cardiomyocytes nibyingenzi mukubungabunga umutima wa fenotype 33,34,35.Mugihe gukanika imashini byakoreshejwe cyane mugukonjesha no gukura kwa hiPSC-CM, ubushakashatsi bwinshi buhebuje buherutse kugerageza gukangurira gukanika ibice byumutima mumico hakoreshejwe imizigo imwe.Ubu bushakashatsi bwerekana ko gupakira 2D uniaxial yamashanyarazi bigira ingaruka nziza kuri phenotype yumutima mugihe cyumuco.Muri ubu bushakashatsi, ibice byumutima byashyizwemo imbaraga za isometric tensile17, umurongo wa auxotonic yipakurura18, cyangwa uruziga rwumutima rwarakozwe hifashishijwe ibitekerezo bya transducer hamwe na drives.Nyamara, ubu buryo bukoresha tissue uniaxial irambuye nta buryo bwiza bwo kubungabunga ibidukikije, bikaviramo guhagarika ingirabuzimafatizo nyinshi z'umutima cyangwa gukabya gukabije kwa gen bifitanye isano no gusubiza bidasanzwe.CTCM yasobanuwe hano itanga imbaraga za 3D zikoresha amashanyarazi yigana umutima wumutima karemano ukurikije igihe cyizunguruka no kurambura umubiri (kurambura 25%, systole 40%, diastole 60%, no gukubitwa 72 kumunota).Nubwo uku gukurura ibice bitatu gusa byonyine bidahagije kugirango uburinganire bwinyama bugume, guhuza imbaraga zo gusetsa no gukanika hakoreshejwe T3 / Dex birasabwa kugirango bikomeze bihagije ubuzima bwimikorere, imikorere, nubunyangamugayo.
Ibintu bisetsa bigira uruhare runini muguhindura umutima ukuze.Ibi byagaragaye mubushakashatsi bwa HiPS-CM aho T3 na Dex byongewe mubitangazamakuru byumuco kugirango byihute gukura kwakagari.T3 irashobora guhindura ubwikorezi bwa aside amine, isukari na calcium mubice byose bigize selile36.Byongeye kandi, T3 iteza imbere imvugo ya MHC-α hamwe no kugabanuka kwa MHC-β, iteza imbere gushiraho myofibrile yihuta muri cardiomyocytes ikuze ugereranije na myofibrile itinda buhoro buhoro muri CM.Kubura T3 kubarwayi ba hypotherideide bivamo gutakaza imitsi ya myofibrillar nigabanuka ryiterambere ryijwi37.Dex ikora kuri reseptor ya glucocorticoid kandi yerekanwe ko yongerera myocardial kwandura mumitima yangiritse;38 iri terambere ritekereza ko rifitanye isano ningaruka kuri calcium yo kubitsa-kwinjiza (SOCE) 39,40.Byongeye kandi, Dex ihuza abayakira, itera igisubizo cyagutse cyo mu nda kigabanya imikorere yumubiri no gutwika30.
Ibisubizo byacu byerekana ko gukangura umubiri (MS) byateje imbere imikorere yumuco muri rusange ugereranije na Ctrl, ariko ntibyashoboye gukomeza kubaho neza, uburinganire bwimiterere, hamwe numutima wumutima muminsi 12 mumuco.Ugereranije na Ctrl, hiyongereyeho T3 na Dex kumico ya CTCM (MT) byateje imbere ubuzima kandi bikomeza imyirondoro isa, ubunyangamugayo, hamwe nibikorwa bya metabolike hamwe nuduce twumutima mushya muminsi 12.Byongeye kandi, mugucunga urwego rwo kurambura tissue, moderi ya hyperextension-iterwa na hypertrophy yumutima yakozwe hakoreshejwe STCM, yerekana uburyo bwinshi bwa sisitemu ya STCM.Twabibutsa ko nubwo kuvugurura umutima hamwe na fibrosis mubisanzwe birimo ingingo zidafite ingirabuzimafatizo zifite uruzinduko zishobora gutanga cytokine ikwiye kimwe na fagocytose hamwe nibindi bintu bivugurura, ibice byumutima birashobora kwigana inzira ya fibrotic mugusubiza ibibazo byihungabana.muri myofibroblast.Ibi byasuzumwe mbere muriki cyitegererezo cyumutima.Twabibutsa ko ibipimo bya CTCM bishobora guhindurwa muguhindura umuvuduko / amashanyarazi amplitude hamwe ninshuro kugirango bigereranye ibintu byinshi nka tachycardia, bradycardia, hamwe nubufasha bwikwirakwizwa ryumutima (umutima udapakurura umutima).Ibi bituma sisitemu yinjira muburyo bwo gupima ibiyobyabwenge.Ubushobozi bwa CTCM bwo kwerekana urugero rwinshi rwumutima-hypertrophy yumutima itanga inzira yo kugerageza ubu buryo bwo kuvura bwihariye.Mu gusoza, ubu bushakashatsi bwerekana ko kurambura imashini no gukurura urwenya ari ingenzi mu gukomeza umuco w’ibice byumutima.
Nubwo amakuru yatanzwe hano yerekana ko CTCM ari urubuga rutanga ikizere cyo kwerekana myocardium idahwitse, ubu buryo bwumuco bufite aho bugarukira.Intambamyi nyamukuru yumuco wa CTCM nuko ishyiraho imbaraga zihoraho zumukanishi ku bice, bikabuza ubushobozi bwo gukurikirana byimazeyo kugabanuka kwimitsi yumutima muri buri cyiciro.Byongeye kandi, bitewe nubunini buke bwibice byumutima (7 mm), ubushobozi bwo gusuzuma imikorere ya systolike hanze yimikorere yumuco ukoresheje ibyuma byimbaraga gakondo ni bike.Muri iki gihe cyandikishijwe intoki, twatsinze igice iyi mbogamizi dusuzuma voltage optique nkikimenyetso cyimikorere yamasezerano.Ariko, iyi mbogamizi izakenera akazi gakomeye kandi irashobora gukemurwa mugihe kizaza mugutangiza uburyo bwo kugenzura neza imikorere yimitima yumutima mumuco, nko gushushanya optique ukoresheje calcium hamwe n amarangi yangiza-voltage.Indi mbogamizi ya CTCM nuko moderi ikora idakoresha imbaraga za physiologique (preload na afterload).Muri CTCM, igitutu cyatewe muburyo butandukanye bwo kubyara 25% bya physiologique muri diastole (kurambura byuzuye) na systole (uburebure bwo kugabanuka mugihe cyo gukurura amashanyarazi) mubice binini cyane.Iyi mbogamizi igomba gukurwaho mugihe kizaza cya CTCM nigitutu gihagije kumitsi yumutima kumpande zombi no gukoresha umubano nyawo wumuvuduko ukabije ubera mubyumba byumutima.
Kurenza urugero-guterwa no kuvugurura ivugwa muri iyi nyandiko yandikishijwe intoki bigarukira gusa ku kwigana ibimenyetso bya hypertrophique hyperstretch.Rero, iyi moderi irashobora gufasha mukwiga kurambura-guterwa na hypertrophique yerekana ibimenyetso bidakenewe ibintu bisekeje cyangwa imitsi (bitabaho muri iyi sisitemu).Iyindi nyigisho irakenewe kugirango ubwiyongere bwa CTCM bugerweho, kurugero, gufatanya ningirabuzimafatizo, gukwirakwiza plasma zitera urwenya, hamwe n’imbere iyo gufatanya n’ingirabuzimafatizo bizamura uburyo bwo kwerekana indwara hamwe na CTCM.
Ingurube 13 zakoreshejwe muri ubu bushakashatsi.Inzira zose z’inyamaswa zakozwe hakurikijwe amabwiriza y’inzego kandi zemejwe na komite ishinzwe kwita ku nyamaswa n’imikoreshereze ya kaminuza ya Louisville.Aortic arch yarafunzwe kandi umutima washyizwemo 1 L ya kardioplegia ya sterile (110 mM NaCl, 1,2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 U / mL heparin, pH kugeza kuri 7.4); imitima yabitswe mubisubizo bikonje byumutima bikonje kugeza bijyanwe muri laboratoire kurubura ubusanzwe <10 min. imitima yabitswe mubisubizo bikonje byumutima bikonje kugeza bijyanwe muri laboratoire kurubura ubusanzwe <10 min. сердца хранили в ледяном кардиоп огическом растворе до сировировки в лабораторию на льду, что обонно занимает <10 мин. imitima yabitswe mubukonje bukonje bwumutima kugeza igihe cyoherezwa muri laboratoire kurubura, ubusanzwe bifata <10 min.将 心脏 保存 在 冰冷 的 心脏停搏 液 中 直到 冰上 10 10 10 <10 分钟。将 心脏 保存 在 冰冷 的 心脏停搏 液 中 直到 冰上 10 10 10 <10 分钟。 Держите сердца в ледяной кардиоп огии до матировки в лабораторию на льду, обоно <10 мин. Gumana imitima kuri ice cardioplegia kugeza ujyanwa muri laboratoire kurubura, mubisanzwe <10 min.
Igikoresho cya CTCM cyakozwe muri software ya SolidWorks ifashwa na mudasobwa (CAD).Ibyumba byumuco, abitandukanya nibyumba byo mu kirere bikozwe muri plastiki ya acrylic ya CNC.Impeta yinyuma ya 7mm ya diametre ikozwe muri polyethylene yuzuye (HDPE) hagati kandi ifite o-ring groove kugirango yakire silicone o-impeta ikoreshwa mu gufunga itangazamakuru munsi.Indimu ya silika yoroheje itandukanya icyumba cyumuco nicyapa cyo gutandukana.Indwara ya silicone ni laser yaciwe kuva 0.02 ″ urupapuro rwa silicone yuzuye kandi ifite ubukana bwa 35A.Hasi ya silicone yo hepfo no hejuru ni laser yaciwe kuva 1/16 ″ umubyimba wa silicone kandi ufite ubukana bwa 50A.316L ibyuma bitagira umuyonga hamwe nutubuto twamababa bikoreshwa mugukomeza umurongo no gukora kashe yumuyaga.
Ikibaho cyabugenewe cyacapwe (PCB) cyashizweho kugirango gihuze na sisitemu ya C-PACE-EM.Imashini ya swiss imashini ihuza socket kuri PCB ihujwe na electrode ya grafite ukoresheje insinga z'umuringa zikozwe mu ifeza hamwe n’umuringa wa 0-60 winjijwe muri electrode.Ikibaho cyumuzingo cyacapwe gishyirwa mubifuniko bya printer ya 3D.
Igikoresho cya CTCM kiyobowe na programuable pneumatic actuator (PPD) ikora umuvuduko ukabije wamaraso usa numutima.Mugihe umuvuduko wimbere mucyumba cyumuyaga wiyongera, silicone ya membrane ihindagurika yaguka hejuru, igahatira uburyo munsi yikibanza.Agace ka tissue kazaramburwa nuku kwirukana amazi, bigana kwaguka kwimiterere yumutima mugihe cya diastole.Mugihe cyo kwidagadura, amashanyarazi yakoreshwaga hakoreshejwe electrode ya grafite, yagabanije umuvuduko wicyumba cyumuyaga kandi bigatuma igabanywa ryibice.Imbere mu muyoboro harimo valve ya hemostatike ifite sensor ya pression kugirango umenye umuvuduko uri muri sisitemu.Umuvuduko ukorwa na sensor sensor ikoreshwa mubikusanyamakuru bihujwe na mudasobwa igendanwa.Ibi bituma uhora ukurikirana umuvuduko uri mucyumba cya gaze.Iyo umuvuduko ntarengwa wicyumba wageze (bisanzwe 80 mmHg, 140 mmHg OS), igikoresho cyo gukusanya amakuru cyategetswe kohereza ikimenyetso muri sisitemu ya C-PACE-EM kugirango gitange ibimenyetso bya biphasic voltage ya ms 2, bishyirwa kuri 4 V.
Ibice byumutima byabonetse kandi imiterere yumuco mumariba 6 yakozwe kuburyo bukurikira: Kwimura imitima yasaruwe mu cyombo cyimurirwa mu kayira karimo umutima ukonje (4 ° C.).Umuyaga wibumoso washyizwe hamwe nicyuma cya sterile hanyuma ucamo ibice 1-2 cm3.Utwo turemangingo twa tissue twafatanyaga hamwe nuduce twa tissue hanyuma tugashyirwa mubwogero bwa microtome ya tissue irimo igisubizo cya Tyrode kandi ikomeza okisijeni (3 g / L 2,3-butanedione monooxime (BDM), 140 mM NaCl (8.18 g).), 6 mM KCl (0.447 g), 10 mM D-glucose (1.86 g) mM CaCl2 (1.8 ml 1 M igisubizo), kugeza 1 L ddH2O).Microtome yinyeganyeza yashyizweho kugirango igabanye ibice 300 µm by'ubugari ku nshuro ya 80 Hz, amplitude ya vibration itambitse ya mm 2, hamwe na 0.03 mm / s.Ubwogero bwa tissue bwari buzengurutswe nurubura kugirango igisubizo gikonje kandi ubushyuhe bwagumishijwe kuri 4 ° C.Kwimura ibice bya tissue kuva muri microtome kwiyuhagira mu bwogero bwa incubation burimo umuti wa ogisijeni uhoraho wa Tyrode ku rubura kugeza igihe habonetse ibice bihagije kuri plaque imwe.Ku mico ya transwell, ibice bya tissue byashyizwe kuri sterile ya mm 6 yubugari bwa polyurethane hanyuma bigashyirwa muri ml 6 yuburyo bwiza (199 hagati, 1x ITS inyongera, 10% FBS, 5 ng / ml VEGF, 10 ng / ml FGF-alkaline na 2X antibiotic-antifungal).Gukangura amashanyarazi (10 V, inshuro 1.2 Hz) byashyizwe mubice byinyama binyuze muri C-Pace.Kubijyanye na TD, T3 nshya na Dex byongewe kuri 100 nM na 1 μM kuri buri mpinduka ziciriritse.Ikigereranyo cyuzuyemo ogisijeni mbere yo gusimburwa inshuro 3 kumunsi.Ibice by'imyenda byatejwe imbere muri incubator kuri 37 ° C na 5% CO2.
Ku mico ya CTCM, ibice byama tissue byashyizwe kumurongo wacapwe wa 3D mugikoresho cya Petri kirimo igisubizo cya Tyrode cyahinduwe.Igikoresho cyagenewe kongera ubunini bwigice cyumutima kuri 25% yubuso bwimpeta.Ibi bikorwa kugirango ibice byumutima bitarambura nyuma yo kwimurwa bivuye mumuti wa Tyrode mukigereranyo no mugihe cya diastole.Ukoresheje kole ya histoacrylic, ibice 300 µm byashyizwe kumurongo wimpeta ya mm 7 ya diameter.Nyuma yo guhuza ibice bya tissue kumpeta yingoboka, gabanya ibice byinyama hanyuma ushireho ibice byinyuma byongeye gusubira mubwogero bwumuti wa Tyrode kurubura (4 ° C) kugeza igihe hateguwe ibice bihagije kubikoresho bimwe.Igihe cyose cyo gutunganya ibikoresho byose ntigomba kurenza amasaha 2.Nyuma yuko ibice 6 byimyenda bifatanye nimpeta zabo, igikoresho cya CTCM cyarateranijwe.Urugereko rwumuco wa CTCM rwujujwe mbere na 21 ml yabanjirije okisijeni.Hindura ibice bya tissue mubyumba byumuco hanyuma ukureho witonze ibyuka byose hamwe na pipeti.Igice cya tissue noneho kiyobora mu mwobo hanyuma ugakanda buhoro buhoro ahantu.Hanyuma, shyira capide ya electrode kubikoresho hanyuma wohereze igikoresho muri incubator.Noneho huza CTCM numuyoboro wikirere na sisitemu ya C-PACE-EM.Pneumatic actuator irakinguka na valve yumuyaga ikingura CTCM.Sisitemu ya C-PACE-EM yashyizweho kugirango itange 4 V kuri 1.2 Hz mugihe cya biphasic igenda kuri ms 2.Ikigereranyo cyahinduwe kabiri kumunsi kandi electrode yahinduwe rimwe kumunsi kugirango birinde gukusanya grafite kuri electrode.Nibiba ngombwa, ibice byinyama birashobora gukurwa mumariba yumuco kugirango birukane umwuka mubi ushobora kuba waguye munsi yabyo.Kubijyanye no kuvura MT, T3 / Dex yongeyeho shyashya hamwe nimpinduka ziciriritse hamwe 100 nM T3 na 1 μM Dex.Ibikoresho bya CTCM byatejwe imbere muri incubator kuri 37 ° C na 5% CO2.
Kugirango ubone inzira ndende zo gukata umutima, hashyizweho sisitemu idasanzwe ya kamera.Kamera ya SLR (Canon Rebel T7i, Canon, Tokiyo, Ubuyapani) yakoreshejwe hamwe na Navitar Zoom 7000 18-108mm ya macro lens (Navitar, San Francisco, CA).Kubona amashusho byakorewe mubushyuhe bwicyumba nyuma yo gusimbuza uburyo nuburyo bushya.Kamera ihagaze kuri 51 ° kandi videwo yandikwa kumirongo 30 kumasegonda.Ubwa mbere, porogaramu ifungura isoko (MUSCLEMOTION43) yakoreshejwe hamwe na Image-J kugirango igabanye icyerekezo cyimitima yumutima.Mask yakozwe hifashishijwe MATLAB (MathWorks, Natick, MA, USA) kugirango isobanure uturere dushimishijwe no gukubita ibice byumutima kugirango twirinde urusaku.Intoki zigabanijwe zikoreshwa kumashusho yose muburyo bukurikiranye hanyuma ikanyuzwa kuri plug-in ya MUSCLEMOTION.Imitsi yimitsi ikoresha impuzandengo yuburemere bwa pigiseli muri buri cyiciro kugirango igereranye ingendo yayo ugereranije nurwego rwerekanwe.Amakuru yanditswe, ayungururwa kandi akoreshwa mukugereranya igihe cyinzira no gusuzuma ingirabuzimafatizo mugihe cyumutima.Video yafashwe amajwi yatunganijwe hifashishijwe icyiciro cya mbere cya zeru icyiciro cya sisitemu.Kugirango ugereranye uduce twinshi (peak-to-peak), isesengura rya pe-to-peak ryakozwe kugirango ritandukanye impinga ninkono mu kimenyetso cyanditse.Mubyongeyeho, gusebanya bikorwa hakoreshejwe gahunda ya 6 ya polinomial kugirango ikureho drift.Kode ya porogaramu yatunganijwe muri MATLAB kugirango hamenyekane icyerekezo cyisi yose, igihe cyizunguruka, igihe cyo kuruhuka, nigihe cyo kugabanuka (Code Programme yinyongera 44).
Kubisesengura, dukoresheje videwo imwe yakozwe mugusuzuma uburyo bwo gukanika imashini, twabanje gukurikirana amashusho abiri yerekana impinga yimikorere (hejuru (hejuru) no hasi (hepfo) ingingo zigenda) dukurikije software ya MUSCLEMOTION.Twahise dutandukanya uturere twa tissue hanyuma dushyira muburyo bwa igicucu cya algorithm kumurongo wacitsemo ibice (Ishusho yinyongera. 2a).Tissue yagabanijwe noneho igabanyijemo ibice icumi byo munsi, kandi guhangayikishwa kuri buri buso byabazwe hakoreshejwe ikigereranyo gikurikira: Strain = (Sup-Sdown) / Sdown, aho Sup na Sdown ari intera yimiterere kuva igicucu cyo hejuru no hepfo yigitambara, (Ishusho yinyongera .2b).
Ibice byumutima byakosowe muri 4% paraformaldehyde mumasaha 48.Utugingo ngengabuzima twavanze twabuze umwuma muri 10% na 20% ya sahrose kuri 1 h, hanyuma muri 30% ya sahrose ijoro ryose.Ibice byahise byinjizwamo ubushyuhe bwiza bwo kugabanya ubushyuhe (OCT compound) hanyuma buhoro buhoro bikonjeshwa muri isopentane / kwiyuhagira urubura.Bika OCT ishiramo ibice kuri -80 ° C kugeza gutandukana.Amashusho yateguwe nkibice bifite ubugari bwa 8 mm.
Kugira ngo ukure OCT mu bice byumutima, shyushya amashusho kumurongo ushyushye kuri 95 ° C kuminota 5.Ongeramo ml 1 PBS kuri buri slide hanyuma ushiremo iminota 30 mubushyuhe bwicyumba, hanyuma winjire mubice ushiraho 0.1% Triton-X muri PBS muminota 15 mubushyuhe bwicyumba.Kugirango wirinde antibodiyite zidasanzwe guhuza icyitegererezo, ongeramo ml 1 yumuti wa 3% BSA kumurongo hanyuma ushireho isaha 1 mubushyuhe bwicyumba.BSA yahise ikurwaho hanyuma amashusho yogejwe hamwe na PBS.Shyira buri cyitegererezo hamwe n'ikaramu.Antibodies yibanze (yavuzwe 1: 200 muri 1% BSA) (connexin 43 (Abcam; # AB11370), NFATC4 (Abcam; # AB99431) na troponin-T (Thermo Scientific; 594 (Ubumenyi bwa Thermo;
WGA-Alexa Fluor 555 (Thermo Scientific; # W32464) kuri 5 μg / ml muri PBS yakoreshejwe mugusiga WGA hanyuma igashyirwa mubice byagenwe muminota 30 mubushyuhe bwicyumba.Amashusho yahise yozwa hamwe na PBS hanyuma umukara wa Sudani wongerwa kuri buri slide hanyuma ushiramo iminota 30.Amashusho noneho yogejwe hamwe na PBS hanyuma hongerwaho uburyo bwa vectashield.Amashusho yagaragaye kuri microscope ya Keyence kuri 40x gukuza.
OCT yakuwe mubitegererezo nkuko byasobanuwe haruguru.Nyuma yo gukuraho OCT, shira amashusho mugisubizo cya Bouin ijoro ryose.Amashusho yahise yozwa namazi yatoboye mumasaha 1 hanyuma ashyirwa mumuti wa Bibrich aloe aside fuchsin muminota 10.Hanyuma amashusho yogejwe namazi yatoboye hanyuma ashyirwa mumuti wa 5% fosifomolibdenum / 5% acide fosifungungike muminota 10.Utabanje kwoza, ohereza amashusho mu buryo butaziguye muri aniline yubururu muminota 15.Hanyuma amashusho yogejwe namazi yatoboye hanyuma ashyirwa mumuti wa 1% acide acike muminota 2.Amashusho yumishijwe muri 200 N Ethanol hanyuma yimurirwa kuri xylene.Ibicapo byerekanwe byerekanwe mikorosikopi ya Keyence ifite intego ya 10x.Agace ka Fibrosis kagereranijwe hifashishijwe software ya Keyence Analyser.
CyQUANT ™ MTT Cell Viability Assay (Invitrogen, Carlsbad, CA), catalog numero V13154, ukurikije protocole yuwabikoze hamwe na bimwe byahinduwe.By'umwihariko, igikoni cyo kubaga gifite umurambararo wa mm 6 cyakoreshejwe kugirango ubunini bwa tissue bumwe mu gihe cyo gusesengura MTT.Utudodo twashyizwe ku giti cyarwo ku mariba y’isahani 12 irimo substrate ya MTT ukurikije protocole yuwabikoze.Ibice byashyizwe kuri 37 ° C. mumasaha 3 kandi tissue nzima ihinduranya substrate ya MTT kugirango ikore uruganda rwumutuku.Simbuza igisubizo cya MTT hamwe na ml 1 DMSO hanyuma ushire kuri 37 ° C muminota 15 kugirango ukure formazan yumutuku mubice byumutima.Ingero zavomwe 1:10 muri DMSO muri 96-neza neza isahani yo hepfo hamwe nuburemere bwamabara yisine yapimye kuri 570 nm ukoresheje umusomyi wa Cytation (BioTek).Gusoma byari bisanzwe muburemere bwa buri gice cyumutima.
Ibice byumutima byasimbuwe nibitangazamakuru birimo 1 μCi / ml [5-3H] -glucose (Moravek Biochemicals, Brea, CA, USA) kugirango ikoreshwe glucose nkuko byasobanuwe mbere.Nyuma yamasaha 4 yubushakashatsi, ongeramo 100 µl yikigereranyo mumiyoboro ifunguye microcentrifuge irimo 100 µl ya 0.2 N HCl.Noneho umuyoboro washyizwe mu muyoboro wa scintillation urimo 500 μl ya dH2O kugirango ushire [3H] 2O mu masaha 72 kuri 37 ° C.Noneho kura umuyoboro wa microcentrifuge muri trintillation hanyuma wongeremo ml 10 ya fluid scintillation.Ibara rya Scintillation ryakozwe hakoreshejwe Tri-Carb 2900TR isesengura isesengura ryamazi (Packard Bioscience Company, Meriden, CT, USA).Ikoreshwa rya glucose noneho ryabazwe hitawe ku bikorwa [5-3H] -glucose ibikorwa byihariye, kuringaniza kutuzuye hamwe ninyuma, kugabanuka kwa [5-3H] -kugirango glucose itemewe, hamwe no gukora neza.Ibyatanzwe nibisanzwe mubice byumutima.
Nyuma yo guhuza ibice muri Trizol, RNA yitandukanije nu gice cyumutima ikoresheje Qiagen miRNeasy Micro Kit # 210874 ukurikije protocole yuwabikoze.Gutegura isomero rya RNAsec, gukurikirana no gusesengura amakuru byakozwe ku buryo bukurikira:
1 μg ya RNA kuri sample yakoreshejwe nkibikoresho byo gutangiza isomero rya RNA.Amasomero akurikirana yakozwe hifashishijwe NEBNext UltraTM RNA Isomero ryo Gutegura Isomero rya Illumina (NEB, AMERIKA) ukurikije ibyifuzo byakozwe nuwabikoze, kandi kodegisi yongeweho kurutonde rwibintu kuri buri cyitegererezo.Muri make, mRNA yasukuwe muri RNA yose ikoresheje amasaro ya magneti afatanye na poly-T oligonucleotide.Gucamo ibice bikorwa hifashishijwe ibipimo bihwanye n'ubushyuhe bwo hejuru muri NEBNext First Strand Synthesis Reaction Buffer (5X).Umugozi wambere cDNA washyizwe hamwe ukoresheje primaire ya hexamer na M-MuLV revers transcriptase (RNase H-).Umugozi wa kabiri cDNA noneho ikomatanyirizwa hamwe ikoresheje ADN polymerase ya I na RNase H. Ibisigaye bisigaye bihindurwamo impera zikorwa na exonuclease / polymerase.Nyuma yo guhindurwa kwa 3 ′ iherezo ryigice cya ADN, Adapt ya NEBNext Adapt ifite imiterere yimisatsi ihambiriye kuri yo kugirango itegure kuvanga.Guhitamo cDNA ibice byuburebure bwatoranijwe 150-200 bp.ibice by'isomero byahanaguwe hakoreshejwe sisitemu ya AMPure XP (Beckman Coulter, Beverly, USA).Noneho, 3 μl USER Enzyme (NEB, USA) hamwe nubunini bwatoranijwe cDNA ihujwe na adapt yakoreshejwe muminota 15 kuri 37 ° C hanyuma muminota 5 kuri 95 ° C mbere ya PCR.PCR noneho yakozwe ikoresheje Phusion High-Fidelity ADN polymerase, primers ya PCR rusange, na Index (X) primers.Hanyuma, ibicuruzwa bya PCR byahanaguweho (sisitemu ya AMPure XP) hamwe nubuziranenge bwibitabo byasuzumwe kuri sisitemu ya Agilent Bioanalyzer 2100.Isomero rya cDNA ryahise rikurikiranwa hakoreshejwe Novaseq.Amadosiye yishusho mabi yo muri Illumina yahinduwe mubisomwa bibisi ukoresheje CASAVA Base Calling.Amakuru yimibare abitswe muri dosiye yimiterere ya FASTQ (fq) ikubiyemo urutonde rwasomwe hamwe nimiterere yibanze.Hitamo HISAT2 kugirango uhuze akayunguruzo gakurikiranye gusoma kuri genoside ya Sscrofa11.1.Muri rusange, HISAT2 ishyigikira genome yubunini ubwo aribwo bwose, harimo genome irenze miliyari 4 shingiro, kandi indangagaciro zisanzwe zashyizweho kubintu byinshi.Gutondeka bisomwa muri RNA Seq amakuru arashobora guhuzwa neza ukoresheje HISAT2, sisitemu yihuta kuboneka, hamwe nuburyo bumwe cyangwa bwiza kuruta ubundi buryo.
Ubwinshi bw'inyandiko-mvugo bugaragaza mu buryo butaziguye urwego rwa gene imvugo.Urwego rwimvugo rusuzumwa nubwinshi bwinyandiko-mvugo (kubara bikurikiranye) bifitanye isano na genome cyangwa exons.Umubare wabasomyi uringaniye nurwego rwerekana imiterere ya gene, uburebure bwa gene, hamwe nubujyakuzimu.FPKM (ibice ku gihumbi fatizo fatizo yinyandiko ikurikiranye kuri miriyoni shingiro) byabazwe kandi P-indangagaciro zerekana imvugo zitandukanye zagenwe hakoreshejwe pake ya DESeq2.Twahise tubara igipimo cyo kuvumbura ibinyoma (FDR) kuri buri gaciro P dukoresheje uburyo bwa Benjamini-Hochberg9 dushingiye kubikorwa byubatswe muri R-imikorere "p.kugorora".
RNA yitandukanije nu gice cyumutima yahinduwe kuri cDNA yibanda kuri 200 ng / μl ikoresheje SuperScript IV Vilo Master ivanze na Thermo (Thermo, injangwe no 11756050).Umubare wa RT-PCR wakozwe hifashishijwe Ikoreshwa rya Biosystems Endura Plate Microamp 384-neza neza isahani yerekana neza (Thermo, injangwe. No 4483319) hamwe na microamp optique (Thermo, injangwe no 4311971).Uruvangitirane rwa reaction rwari rugizwe na 5 µl Taqman Byihuta Byihuta Bivanze (Thermo, injangwe # 4444557), 0.5 µl Taqman Primer na 3.5 µl H2O bivanze kuriba.Inzira zisanzwe za qPCR zakozwe kandi indangagaciro za CT zapimwe hifashishijwe Ikoreshwa rya Biosystems Quantstudio 5 igikoresho nyacyo cya PCR (384-neza neza; ibicuruzwa # A28135).Taqman primers yaguzwe muri Thermo (GAPDH (Ss03375629_u1), PARP12 (Ss06908795_m1), PKDCC (Ss06903874_m1), CYGB (Ss06900188_m1), RGL1 (Ss06868890_M1) 33.
Itangazwa rya NT-ProBNP ryasuzumwe hifashishijwe ibikoresho bya NT-ProBNP (ingurube) (Cat. No MBS2086979, MyBioSource) ukurikije protocole yuwabikoze.Muri make, 250 µl ya buri sample hamwe nibisanzwe byongewemo kabiri kuri buri riba.Ako kanya nyuma yo kongeramo icyitegererezo, ongeramo 50 µl ya Assay Reagent A kuri buri riba.Kunyeganyeza witonze isahani hanyuma ushireho kashe.Hanyuma ibinini byashyizwe kuri 37 ° C kumasaha 1.Noneho shaka igisubizo hanyuma ukarabe amariba inshuro 4 hamwe na 350 µl yumuti wa 1X woza, ushizemo igisubizo cyo gukaraba muminota 1-2 buri mwanya.Noneho ongeramo 100 µl ya Assay Reagent B kuri iriba hanyuma ushireho kashe ya plaque.Ikibaho cyajugunywe buhoro kandi gishyirwa kuri 37 ° C mu minota 30.Saba igisubizo hanyuma ukarabe amariba inshuro 5 hamwe na 350 µl ya 1X yo gukaraba.Ongeramo 90 µl yumuti wa substrate kuri buri riba hanyuma ushireho isahani.Shyiramo isahani kuri 37 ° C muminota 10-20.Ongeramo 50 µl Hagarika igisubizo kuri buri riba.Isahani yahise ipimwa hifashishijwe icyapa cya Cytation (BioTek) cyashyizwe kuri 450 nm.
Isesengura ryimbaraga ryakozwe kugirango uhitemo ingano yitsinda rizatanga> 80% imbaraga zo kumenya 10% ihinduka ryuzuye mubintu hamwe na 5% byikosa ryubwoko bwa I. Isesengura ryimbaraga ryakozwe kugirango uhitemo ingano yitsinda rizatanga> 80% imbaraga zo kumenya 10% ihinduka ryuzuye mubintu hamwe na 5% byikosa ryubwoko bwa I. Анз мощности был выполнен для выбора размеров групп, кот кае обеспечат> 80% мощности для обнаружения 10% абсолтного изменения Параметра с 5% частотой чибобок ча I. Isesengura ryimbaraga ryakozwe kugirango uhitemo ingano yitsinda ryatanga> 80% imbaraga zo gutahura 10% byimiterere ihinduka hamwe na 5% yikosa ryubwoko bwa 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 组 大小。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 组 大小。 Был проекты Isesengura ryimbaraga ryakozwe kugirango uhitemo ingano yitsinda ryatanga> 80% imbaraga zo kumenya 10% ihinduka ryimiterere ihindagurika na 5% yubwoko bwikosa.Ibice by'imyenda byatoranijwe mbere yubushakashatsi.Isesengura ryose ryabaye impumyi kandi ingero zashizweho nyuma yamakuru yose amaze gusesengurwa.Porogaramu ya GraphPad Prism (San Diego, CA) yakoreshejwe mu gusesengura imibare yose. Kubarurishamibare ryose, p-agaciro byafatwaga nkibyingenzi kubiciro <0.05. Kubarurishamibare ryose, p-agaciro byafatwaga nkibyingenzi kubiciro <0.05. Для всей стати мати p-значения считались значимыми при значениях <0,05. Kubarurishamibare ryose, p-agaciro byafatwaga nkibyingenzi kubiciro <0.05.对于 所有 统计 数据 ， p 值 在 值 <0.05 时 被 认为 是 显 着 的。对于 所有 统计 数据 ， p 值 在 值 <0.05 时 被 认为 是 显 着 的。 Для всей стати мати p-значения считались значимыми при значениях <0,05. Kubarurishamibare ryose, p-agaciro byafatwaga nkibyingenzi kubiciro <0.05.Ikizamini cya t-umurizo wabanyeshuri cyakorewe kumibare ugereranije 2 gusa.Inzira imwe cyangwa inzira ebyiri ANOVA yakoreshejwe kugirango hamenyekane akamaro hagati yitsinda ryinshi.Mugihe ukora ibizamini bya post hoc, ubugororangingo bwa Tukey bwakoreshejwe kugirango ubare kugereranya byinshi.Amakuru ya RNAsec afite ibitekerezo byihariye byibarurishamibare mugihe ubara FDR na p.kugirango nkuko byasobanuwe mubice byuburyo.
Kubindi bisobanuro kubijyanye nigishushanyo mbonera, reba Raporo yubushakashatsi bwibidukikije ifitanye isano niyi ngingo.