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Pane chikonzero chekushandisa system yakavimbika ye in vitro inogona kudzokorora mamiriro emuviri emwoyo nenzira yakarurama kuti iongororwe mishonga. Kusawanikwa kwemaitiro ekugadzira tissue dzemwoyo wemunhu kwakaita kuti pave nekusanzwisiswa kwemhedzisiro yemishonga yemwoyo. Pano, takagadzira modhi yekugadzirwa kwetissue dzemwoyo (CTCM) iyo inokurudzira zvidimbu zvemoyo nemagetsi uye inotambanudzwa mumuviri panguva ye systolic ne diastolic phases ye cardiac cycle. Mushure memazuva gumi nemaviri ekugadzirwa, nzira iyi yakavandudza zvishoma kushanda kwezvikamu zvemoyo, asi haina kuchengetedza zvizere kusimba kwechimiro chazvo. Naizvozvo, mushure mekuongororwa kwemamorekuru madiki, takaona kuti kuwedzerwa kwe 100 nM triiodothyronine (T3) uye 1 μM dexamethasone (Dex) kune medium yedu kwakachengetedza chimiro chezvikamu kwemazuva gumi nemaviri. Pamwe chete nekurapwa kweT3/Dex, system yeCTCM yakachengetedza ma profiles e transcriptional, kushanda, metabolic activity, uye kusimba kwechimiro padanho rakafanana ne tissue yemoyo mutsva kwemazuva gumi nemaviri. Pamusoro pezvo, kutambanudza zvakanyanya kwenyama yemwoyo mutsika kunokonzera zviratidzo zvemwoyo zvakanyanya, zvichipa humbowo hwekukwanisa kweCTCM kutevedzera mamiriro ezvinhu anokonzerwa nekutambanudza kwemoyo. Mukupedzisa, CTCM inogona kutevedzera mashandiro emuviri uye pathophysiology yemoyo mutsika kwenguva yakareba, zvichiita kuti kuongororwa kwemishonga kuve kwakavimbika.
Pamberi pekutsvagurudza kwekiriniki, masisitimu akavimbika e in vitro anodiwa anogona kuburitsa zvakanaka mamiriro emuviri emwoyo wemunhu. Masisitimu akadaro anofanira kutevedzera kutambanudza kwakachinja kwemagetsi, kurova kwemoyo, uye hunhu hwe electrophysiological. Mhando dzemhuka dzinowanzo shandiswa senzvimbo yekuongorora physiology yemwoyo isina kuvimbika kwakawanda mukuratidza mhedzisiro yemishonga mumoyo wemunhu1,2. Pakupedzisira, Ideal Cardiac Tissue Culture Experimental Model (CTCM) imhando inonyanya kunzwisisika uye yakanangana nekurapa kwakasiyana-siyana uye mishonga, ichidzokorora nemazvo physiology uye pathophysiology yemoyo wemunhu3. Kushaikwa kwesisitimu yakadaro kunoganhurira kuwanikwa kwemishonga mitsva yekutadza kwemoyo4,5 uye kwakaita kuti pave nekukuvadza kwemoyo nemishonga sechikonzero chikuru chekubuda mumusika6.
Mumakore gumi apfuura, mishonga misere isiri yemwoyo yakabviswa kushandiswa mukiriniki nekuti inokonzera kuwedzera kwenguva yeQT zvichikonzera ventricular arrhythmias uye kufa kamwe kamwe7. Saka, pane kudiwa kuri kuwedzera kwemaitiro ekuongorora akavimbika epamberi pekiriniki kuti vaongorore kushanda kwemwoyo uye chepfu. Kushandiswa kwazvino kwe cardiomyocytes dzinobva mumasero emwoyo (hiPS-CM) dzinobva kuvanhu mukuongorora mishonga uye kuyedza chepfu kunopa mhinduro isina kukwana kudambudziko iri. Zvisinei, hunhu hwema hiPS-CM husina kukura uye kushaikwa kwekuomarara kwemasero emwoyo ndizvo zvinotadzisa nzira iyi. Zvidzidzo zvechangobva kuitwa zvakaratidza kuti kuganhurirwa uku kunogona kukundwa nekushandisa early hiPS-CM kugadzira mahydrogel ematishu emwoyo nguva pfupi mushure mekutanga kwekugumburwa kwemuviri uye kuwedzera zvishoma nezvishoma kukurudzira kwemagetsi nekufamba kwenguva. Zvisinei, aya ma microtissue e hiPS-CM haana hunhu hwe electrophysiological uye contractile hwematumbu emwoyo emunhu mukuru. Pamusoro pezvo, nyama yemwoyo yemunhu ine chimiro chakaoma kunzwisisa, chine musanganiswa wakasiyana-siyana wemarudzi akasiyana emasero, kusanganisira masero e endothelial, neurons, uye stromal fibroblasts, zvakabatana nemaseti chaiwo emapuroteni e extracellular matrix. Kusiyana uku kwevasiri ve cardiomyocyte populations11,12,13 mumoyo wemhuka huru idambudziko guru pakugadzira nyama yemwoyo uchishandisa mhando dzemasero ega ega. Izvi zvinosimbisa kukosha kwekugadzira nzira dzekugadzira nyama yemoyo isina kukuvadzwa pasi pemamiriro emuviri uye ezvirwere.
Zvikamu zvakatetepa zvemwoyo wemunhu zvakakura (300 µm) zvakaratidza kuva muenzaniso unovimbisa we myocardium yemunhu isina kukuvadzwa. Nzira iyi inopa mukana wekuwana system yakazara ye3D multicellular yakafanana nenyama yemwoyo yemunhu. Zvisinei, kusvika muna 2019, kushandiswa kwezvikamu zvemoyo zvakakura kwakaganhurirwa nekurarama kwenguva pfupi (maawa makumi maviri nemana) kwetsika. Izvi zvinokonzerwa nezvinhu zvakawanda zvinosanganisira kushaikwa kwekutambanudza kwemuviri-kwenyama, interface yemhepo-yemvura, uye kushandiswa kwemidziyo iri nyore isingatsigire zvinodiwa nenyama yemwoyo. Muna 2019, mapoka akati wandei ekutsvagisa akaratidza kuti kuisa zvinhu zvemakanika mumasisitimu etsika yemoyo kunogona kuwedzera hupenyu hwetsika, kuvandudza kutaura kwemoyo, uye kutevedzera chirwere chemwoyo. Zvidzidzo zviviri zvakanaka 17 na18 zvinoratidza kuti kurodha kwemagetsi kweuniaxial kune mhedzisiro yakanaka pa phenotype yemoyo panguva yetsika. Zvisinei, zvidzidzo izvi hazvina kushandisa dynamic three-dimensional physico-mechanical loading ye cardiac cycle, sezvo zvikamu zvemoyo zvaive zvakazadzwa ne isometric tensile forces 17 kana linear auxotonic loading 18. Nzira idzi dzekutambanudza tissue dzakakonzera kudzvinyirirwa kwemajini mazhinji emwoyo kana kuti kuwanda kwemajini ane chekuita nemhinduro dzisina kujairika dzekutambanudza. Zvinonyanya kukosha, Pitoulis et al. 19 vakagadzira nzira yekuvandudza tsinga dzemwoyo ine simba yekuvakazve tsinga dzemwoyo vachishandisa force transducer feedback uye tension drives. Kunyangwe iyi sisitimu ichibvumira kutevedzera kwakarurama kwe in vitro cardiac cycle modelling, kuomarara uye kuderera kwenzira iyi kunoderedza kushandiswa kweiyi sisitimu. Rabhoritari yedu ichangobva kugadzira sisitimu yekuvandudza tsinga ichishandisa electrical stimulation uye optimized medium kuchengetedza kushanda kwezvikamu zvenyama yenguruve nemoyo wemunhu kwemazuva anosvika matanhatu.
Muchinyorwa chazvino, tinotsanangura muenzaniso wetsika dzemwoyo (CTCM) tichishandisa zvikamu zvemoyo wenguruve zvinosanganisira humoral cues kuti tidzokorore physiology yemwoyo ine mativi matatu uye pathophysiological distension panguva yekufamba kwemoyo. Iyi CTCM inogona kuwedzera kururama kwekufanotaura kwemishonga isati yasvika pakiriniki kusvika padanho risati ramboonekwa nekupa hurongwa hwemwoyo hunoshanda, hwepakati-pakati hunotevedzera physiology/pathophysiology yemwoyo wemhuka dzinoyamwisa kuti zviongororwe mishonga isati yasvika pakiriniki.
Zviratidzo zveHemodynamic mechanical zvinoita basa rakakosha mukuchengetedza mashandiro emwoyo mu vitro 22,23,24. Muchinyorwa chazvino, takagadzira CTCM (Mufananidzo 1a) inogona kutevedzera mamiriro emwoyo wemunhu mukuru nekukonzera kukurudzirwa kwemagetsi uye kwemakemikari pamafambiro emuviri (1.2 Hz, 72 beats paminiti). Kuti tidzivise kutambanudza kwenyama zvakanyanya panguva yediastole, mudziyo wekudhinda we3D wakashandiswa kuwedzera saizi yenyama ne25% (Mufananidzo 1b). Kufamba kwemagetsi kunokonzerwa neC-PACE system kwakarongerwa kuti kutange 100 ms systole isati yatanga uchishandisa sisitimu yekuwana data kuti ibudise zvizere kutenderera kwemoyo. Sisitimu yekurima tissue inoshandisa actuator inogona kurongwa (LB Engineering, Germany) kuti iwedzere flexible silicone membrane kuti ikonzere kukura kwezvidimbu zvemoyo mukamuri repamusoro. Sisitimu iyi yakabatana netambo yemhepo yekunze kuburikidza ne pressure transducer, izvo zvakaita kuti zvikwanisike kugadzirisa kumanikidzwa nemazvo (± 1 mmHg) nenguva (± 1 ms) (Mufananidzo 1c).
a Batanidza chikamu chetishu kune rin'i yekutsigira ye7 mm, inoratidzwa nebhuruu, mukati mekamuri yekusimudzira yemudziyo. Kamuri yekusimudzira yakaparadzaniswa nekamuri yemhepo nekamera katete kesilicone kanochinjika. Isa gasket pakati pekamuri yega yega kudzivirira kubuda kwemvura. Chivharo chemudziyo chine ma electrode e graphite anopa kukurudzira kwemagetsi. b Chiratidzo chechimiro chemudziyo mukuru wetishu, rin'i yekutungamira uye rin'i yekutsigira. Zvikamu zvetishu (brown) zvinoiswa pamudziyo mukuru nerin'i yekutungamira yakaiswa mugoronga riri pamucheto wekunze wemudziyo. Uchishandisa gwara, isa nokungwarira rin'i yekutsigira yakafukidzwa ne tissue acrylic adhesive pamusoro pechikamu chetishu yemwoyo. c Girafu inoratidza nguva yekukurudzira kwemagetsi sebasa rekumanikidzwa kwekamuri yemhepo inodzorwa ne programmable pneumatic actuator (PPD). Mudziyo wekuwana data wakashandiswa kuwiriranisa kukurudzira kwemagetsi uchishandisa masensa ekumanikidza. Kana kumanikidzwa mukamuri yekusimudzira kwasvika pamuganho wakaiswa, chiratidzo chepulse chinotumirwa kuC-PACE-EM kuti chitange kukurudzira kwemagetsi. d Mufananidzo weCTCM ina dzakaiswa pasherufu ye incubator. Midziyo mina yakabatana nePPD imwe chete kuburikidza necircuit yepneumatic, uye masensa ekumanikidza anoiswa muvharuvhu yehemostatic kuti atarise kumanikidzwa kuri mucircuit yepneumatic. Mudziyo wega wega une zvikamu zvitanhatu zvetishu.
Tichishandisa muchina mumwe chete wekufema, takakwanisa kudzora michina mina yeCTCM, imwe neimwe yaigona kutakura zvikamu zvitanhatu zvenyama (Mufananidzo 1d). MuCTCM, kumanikidzwa kwemhepo mukamuri yemhepo kunoshandurwa kuita kumanikidzwa kwakafanana mukamuri yemvura uye kunokonzera kukura kwechikamu chemoyo (Mufananidzo 2a neSupplementary Movie 1). Kuongororwa kwekutambanudza kwenyama pa80 mm Hg. Art. kwakaratidza kutambanudza zvikamu zvenyama ne25% (Mufananidzo 2b). Kutambanudza uku kwakaratidzwa kuti kunoenderana nehurefu hwesarcomere yemuviri hwe2.2–2.3 µm hwechikamu chemoyo chakajairika17,19,25. Kufamba kwenyama kwakaongororwa uchishandisa zvigadziriso zvekamera yakasarudzika (Mufananidzo 1 wekuwedzera). Amplitude uye kumhanya kwekufamba kwenyama (Mufananidzo 2c, d) zvinoenderana nekutambanudza panguva yekufamba kwemoyo nenguva panguva yesystole nediastole (Mufananidzo 2b). Kutambanudza uye kumhanya kwenyama yemoyo panguva yekudzikira uye kuzorora kwakaramba kuripo kwemazuva gumi nemaviri mukukudziridza (Mufananidzo 2f). Kuti tiongorore mhedzisiro yekukurudzira kwemagetsi pakugumira panguva yekurima, takagadzira nzira yekuona kuremara kuri kushanda tichishandisa shading algorithm (Supplementary Fig. 2a,b) uye takakwanisa kusiyanisa kuremara kuripo nekukurudzira kwemagetsi uye kusina. Chikamu chimwe chete chemoyo (Fig. 2f). Munzvimbo inofambiswa yekucheka (R6-9), voltage panguva yekukurudzira kwemagetsi yaive yakakwira ne20% kupfuura pasina kukurudzira kwemagetsi, izvo zvinoratidza mupiro wekukurudzira kwemagetsi kubasa rekubata.
Zviratidzo zvinoratidza kumanikidzwa kwemukamuri yemhepo, kumanikidzwa kwemukamuri yemvura, uye kuyerwa kwekufamba kwenyama zvinosimbisa kuti kumanikidzwa kwemukamuri kunochinja kumanikidzwa kwemukamuri yemvura, zvichikonzera kufamba kwakafanana kwechidimbu chenyama. b Zviratidzo zvinoratidza kutambanudza kwezana (bhuruu) kwezvikamu zvenyama zvinoenderana nekutambanudza kwezana (orenji). c Kufamba kwakayerwa kwechidimbu chemoyo kunoenderana nekumhanya kwakayerwa kwekufamba. (d) Nzira dzinoratidza kufamba kwekufamba kwemukamuri (mutsetse webhuruu) uye kumhanya (mutsetse une madota eorenji) muchidimbu chemoyo. e Kuyera nguva yekutenderera (n = zvidimbu 19 paboka, kubva kunguruve dzakasiyana), nguva yekubata (n = zvidimbu 19 paboka), nguva yekuzorora (n = zvidimbu 19 paboka, kubva kunguruve dzakasiyana), kufamba kwenyama (n = 25). zvidimbu)/boka kubva kunguruve dzakasiyana), kumhanya kwepakati kwesystolic (n = 24(D0), zvidimbu 25(D12)/boka kubva kunguruve dzakasiyana) uye mwero wekuzorora kwepamusoro (n = 24(D0), zvidimbu 25(D12)/boka kubva kunguruve dzakasiyana). T-test yeMudzidzi ine miswe miviri haina kuratidza mutsauko wakakosha mune chero paramita. f Kuongororwa kwemhando yetsinga dzezvikamu zvenyama zvine (tsvuku) uye zvisina (bhuruu) kukurudzira kwemagetsi, nzvimbo gumi dzematunhu dzezvikamu zvenyama kubva muchikamu chimwe chete. Mapaneru epasi anoratidza huwandu hwemusiyano weperesenti mukumanikidzwa muzvikamu zvenyama zvine uye zvisina kukurudzira kwemagetsi munzvimbo gumi kubva muzvikamu zvakasiyana. (n = zvidimbu zvisere/boka kubva kunguruve dzakasiyana, bvunzo yeT-test yeMudzidzi ine miswe miviri inoitwa; ****p < 0.0001, **p < 0.01, *p < 0.05). (n = zvidimbu zvisere/boka kubva kunguruve dzakasiyana, bvunzo yeT-test yeMudzidzi ine miswe miviri inoitwa; ****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 = zvikamu 8/boka kubva kunguruve dzakasiyana, bvunzo yeT yeMudzidzi ine miswe miviri; ****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 = zvikamu 8/boka, kubva kunguruve dzakasiyana, bvunzo yeT yeMudzidzi ine miswe miviri; ****p<0.0001, **p<0.01, *p<0.05).Mitsetse yekukanganisa inomiririra avhareji ± kutsauka kwakajairika.
Muhurongwa hwedu hwekare hwekusimudzira moyo hwe static biomimetic [20, 21], takachengetedza kushanda, kushanda, uye kusimba kwechimiro chezvidimbu zvemoyo kwemazuva matanhatu nekushandisa electrical stimulation uye kugadzirisa medium composition. Zvisinei, mushure memazuva gumi, nhamba idzi dzakadzikira zvakanyanya. Tichataura nezvezvikamu zvakarimwa mu static biomimetic culture system yedu yekare 20, 21 control conditions (Ctrl) uye tichashandisa medium yedu yakagadziridzwa kare se MC conditions uye culture pasi pe simultaneous mechanical and electrical stimulation (CTCM). inonzi . Kutanga, takaona kuti mechanical stimulation isina electrical stimulation yakanga isina kukwana kuchengetedza tissue liability kwemazuva matanhatu (Supplementary Fig. 3a,b). Zvinofadza kuti, nekuunzwa kwe physio-mechanical uye electrical stimulation uchishandisa STCM, kushanda kwezvikamu zvemoyo zvemazuva gumi nemaviri kwakaramba kwakafanana nekwezvikamu zvemoyo mutsva pasi peMS conditions, asi kwete pasi peCtrl conditions, sezvakaratidzwa ne MTT analysis (Fig. 1). 3a). Izvi zvinoratidza kuti mechanical stimulation uye simulation ye cardiac cycle zvinogona kuchengetedza zvikamu zvenyama zvichirarama kwenguva yakareba kaviri pane zvakataurwa mu static culture system yedu yapfuura. Zvisinei, kuongororwa kwehunhu hwezvikamu zvenyama nekushandisa immunolabeling ye cardiac troponin T ne connexin 43 kwakaratidza kuti connexin 43 expression yaive yakakwira zvakanyanya mu MC tissues pazuva rechi12 pane mu controls pazuva rimwe chete. Zvisinei, uniform connexin 43 expression uye Z-disc formation hazvina kuchengetedzwa zvizere (Mufananidzo 3b). Tinoshandisa artificial intelligence (AI) framework kuti tiverenge hunhu hwe tissue structural integrity26, mufananidzo-based deep learning pipeline yakavakirwa pa troponin-T uye connexin staining43 kuti tiverenge otomatiki hunhu hwe structural integrity uye fluorescence ye heart slices maererano nesimba re localization. Iyi nzira inoshandisa Convolutional Neural Network (CNN) uye deep learning framework kuti tiverenge zvakavimbika hunhu hwe cardiac tissue nenzira otomatiki uye isina rusaruro, sezvakatsanangurwa mu reference. 26. MC tissue yakaratidza kuvandudzika kwehunhu kusvika pazuva 0 zvichienzaniswa ne static control sections. Pamusoro pezvo, kutsvukirira kwemavara etrichrome kwaMasson kwakaratidza huwandu hwakaderera hwe fibrosis pasi pezvirwere zveMS zvichienzaniswa nemamiriro ekudzora pazuva rechi12 rekurera (Mufananidzo 3c). Kunyange zvazvo CTCM yakawedzera kushanda kwezvikamu zvenyama yemoyo pazuva rechi12 kusvika padanho rakafanana nerenyama yemoyo mutsva, hazvina kuvandudza zvakanyanya kusimba kwezvikamu zvemoyo.
Girafu yebha inoratidza kuyera kweMTT kurarama kwezvidimbu zvemoyo zvitsva (D0) kana kukura kwezvidimbu zvemoyo kwemazuva gumi nemaviri kungave mukukudziridza kwemuviri (D12 Ctrl) kana muCTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) zvidimbu/boka kubva kunguruve dzakasiyana, nzira imwe chete inoitwa bvunzo yeANOVA; ####p < 0.0001 zvichienzaniswa neD0 uye **p < 0.01 zvichienzaniswa neD12 Ctrl). Girafu yebha inoratidza huwandu hweMTT inoshanda zvakanaka yezvidimbu zvemoyo zvitsva (D0) kana kuti zvidimbu zvemoyo kwemazuva gumi nemaviri kungave muchimiro chestatic culture (D12 Ctrl) kana muCTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) zvidimbu/boka kubva kunguruve dzakasiyana, nzira imwe chete inoitwa bvunzo yeANOVA; ####p < 0.0001 zvichienzaniswa neD0 uye **p < 0.01 zvichienzaniswa neD12 Ctrl).histogram inoratidza huwandu hwekushanda kwezvikamu zvemwoyo zvitsva zveMTT (D0) kana tsika yezvikamu zvemoyo kwemazuva gumi nemaviri mukuumbwa kwemasero asina kusimba (D12 control) kana CTCM (D12 MC) (n = 18 (D0), 15 (D12 control). ) ), zvikamu gumi nezviviri (D12 MC) kubva kunguruve dzakasiyana, bvunzo yeANOVA inoitwa nenzira imwe chete;####p < 0,0001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). ####p < 0.0001 zvichienzaniswa naD0 uye **p < 0.01 zvichienzaniswa naD12 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) 或片片(D0) 18 (D0), 15 (D12 Ctrl) 新鲜心脏切片(D0) 或天的MTT 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 <0.00D0 → Ctrl 1,000 < 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) 新鲜心脏切片(D0) 中新鲜心脏切片(D0)2) (D12 MC)切片/组，进行单向ANOVA 测试；与D0 相比，####p <0.0001，与D12 Ctrl 相比，**p.)histogram inoratidza huwandu hweMTT viability muzvikamu zvemwoyo mutsva (D0) kana zvikamu zvemoyo zvakarimwa kwemazuva gumi nemaviri mukusimudzirwa kwemuviri (D12 control) kana CTCM (D12 MC) (n = 18 (D0), 15 (D12 control)), 12 (D12 MC) zvikamu/boka kubva kunguruve dzakasiyana, bvunzo yeANOVA yenzira imwe chete;####p < 0,0001 по сравнению с D0, **p < 0,01 по сравнению с D12 Ctrl). ####p < 0.0001 zvichienzaniswa naD0, **p < 0.01 zvichienzaniswa naD12 Ctrl).b Troponin-T (girini), connexin 43 (tsvuku) uye DAPI (bhuruu) muzvikamu zvemwoyo zvitsva (D0) kana zvikamu zvemoyo zvakarimwa pasi pemamiriro ezvinhu asingachinji (Ctrl) kana mamiriro eCTCM (MC) kwemazuva gumi nemaviri) emifananidzo inomiririra immunofluorescence (chikero chisina chinhu = 100 µm). Kuyerwa kwehungwaru hwekugadzira kwehunhu hwemoyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) zvidimbu/boka rimwe nerimwe kubva kunguruve dzakasiyana, bvunzo yeANOVA yenzira imwe chete inoitwa; ####p < 0.0001 zvichienzaniswa naD0 uye ****p < 0.0001 zvichienzaniswa naD12 Ctrl). Kuyera hungwaru hwekugadzira hunhu hwemoyo (n = 7 (D0), 7 (D12 Ctrl), zvidimbu zvishanu (D12 MC) kubva muboka rimwe nerimwe kubva munguruve dzakasiyana, bvunzo yeANOVA inoitwa nenzira imwe chete; ####p < 0.0001 zvichienzaniswa naD0 uye ****p < 0.0001 zvichienzaniswa naD12 Ctrl). Количественная оценка структурной целостности сердечной ткани искусственным интеллектом (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) сруправи/ проводится однофакторный тест ANOVA; Kuyera kusimba kwechimiro chenyama yemwoyo kuburikidza nepfungwa dzekugadzira (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) zvikamu/mapoka kubva kunguruve dzakasiyana, bvunzo yeANOVA yakaitwa nenzira imwe chete; ####p < 0.0001 vs. naD0 uye ****p < 0.0001 zvichienzaniswa naD12 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) zvimedu/boka imwe neimwe yenguruve yakasiyana, nzira imwe ANOVA bvunzo;##0#0D0D010.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) zvimedu/boka imwe neimwe yenguruve dzakasiyana, nzira imwe ANOVA bvunzo;##0#0D0D **** **** <0.0001 与D12 Ctrl 相比）. Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC/зрка) сказал разных свиней, односторонний тест ANOVA <0,0001 vs. D0 Для сравнения ****p <0,0001 по сравнению с D12 Ctrl). Uchenjeri hwekugadzira kuti uone kusimba kwemuviri wemwoyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) zvikamu/boka rega rega renguruve dzakasiyana, bvunzo yeANOVA yenzira imwe chete; ####p<0.0001 vs .D0 Kuenzanisa ****p < 0.0001 zvichienzaniswa neD12 Ctrl). c Mifananidzo inomiririra (kuruboshwe) uye chiyero (kurudyi) chezvidimbu zvemoyo zvakapendwa neMasson's trichrome stain (Scale bare = 500 µm) (n = zvidimbu gumi/boka rimwe nerimwe kubva kunguruve dzakasiyana, bvunzo yeANOVA yenzira imwe chete inoitwa; ####p < 0.0001 zvichienzaniswa naD0 uye ***p < 0.001 zvichienzaniswa naD12 Ctrl). c Mifananidzo inomiririra (kuruboshwe) uye chiyero (kurudyi) chezvidimbu zvemoyo zvakapendwa neMasson's trichrome stain (Scale bare = 500 µm) (n = zvidimbu gumi/boka rimwe nerimwe kubva kunguruve dzakasiyana, bvunzo yeANOVA yenzira imwe chete inoitwa; #### p < 0.0001 zvichienzaniswa naD0 uye ***p < 0.001 zvichienzaniswa naD12 Ctrl). c Репрезентативные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красителем Масязко = мкм) (n = 10 срезов/группу от разных свиней, выполняется односторонний тест ANOVA; c Mifananidzo inomiririra (kuruboshwe) uye kuyera (kurudyi) kwezvikamu zvemoyo zvakasvibiswa netsvina yeMasson's trichrome (chikero chisina kuvharwa = 500 µm) (n = zvikamu gumi/boka kubva kunguruve dzakasiyana, bvunzo yeANOVA yakaitwa nenzira imwe chete; #### p < 0 .0001 zvichienzaniswa neD0 uye ***p < 0.001 zvichienzaniswa neD12 Ctrl). 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 µm） 500个切片/组，每组來自不同的猪，进行单向ANOVA 测试； 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 裸尺度 裸尺度 裸尺度 裸尺度 裸尺庰 裸尺庰裸尺尺庰 （左 左） 量化= 500 µm） （n = 10 个 切片 组 每 來自 不同 猪 , 进行 单向 单向 Anova 测试；#### D0 <0.0. <0.001 D12 Ctrl dhizaini. c Репрезентативные изображения (слева) и количественный анализ (справа) срезов сердца, окрашенных трихромным красителем Массом Массоя =ш (n = 10 срезов/группа, каждый от другой свиньи, протестировано с помощью однофакторного дисперсионного анализа ;####p <0,000,000 сравнению с D12 Ctrl). c Mifananidzo inomiririra (kuruboshwe) uye huwandu (kurudyi) hwezvikamu zvemoyo zvakapendwa neMasson's trichrome stain (isina chinhu = 500 µm) (n = zvikamu gumi/boka, chimwe nechimwe chichibva kune imwe nguruve yakasiyana, yakaedzwa nekuongorora musiyano wenzira imwe chete ;### # p < 0.0001 zvichienzaniswa naD0, ***p < 0.001 zvichienzaniswa naD12 Ctrl).Mitsetse yekukanganisa inomiririra avhareji ± kutsauka kwakajairika.
Takafungidzira kuti nekuwedzera mamorekuru madiki panzvimbo yekurera, cardiomyocyte integrity inogona kuvandudzwa uye kukura kwefibrosis kuderedzwa panguva yekurera kweCTCM. Saka takaongorora mamorekuru madiki tichishandisa static control cultures20,21 nekuda kwehuwandu hushoma hwezvinhu zvinovhiringidza. Dexamethasone (Dex), triiodothyronine (T3), uye SB431542 (SB) zvakasarudzwa pakuongorora uku. Mamorekuru madiki aya akamboshandiswa mu hiPSC-CM cultures kukurudzira kukura kwe cardiomyocytes nekuwedzera urefu hwe sarcomere, T-tubules, uye conduction velocity. Pamusoro pezvo, ese Dex (glucocorticoid) neSB zvinozivikanwa nekudzora kuzvimba29,30. Saka, takaedza kana kuiswa kweimwe kana musanganiswa wemamorekuru madiki aya kwaizovandudza structural integrity yezvikamu zvemoyo. Pakuongorora kwekutanga, dose yekombuta yega yega yakasarudzwa zvichibva pahuwandu hunowanzo shandiswa mu cell culture models (1 μM Dex27, 100 nM T327, uye 2.5 μM SB31). Mushure memazuva gumi nemaviri ekurera, kusanganiswa kweT3 neDex kwakaita kuti pave nekugadzikana kwakanaka kwechimiro chemwoyo uye kugadziridzwa kushoma kwefibrous (Zvifananidzo Zvekuwedzera 4 ne5). Pamusoro pezvo, kushandiswa kwezvikamu zviviri kana zviviri zveT3 neDex kwakakonzera migumisiro yakaipa zvichienzaniswa nezvikamu zvakajairwa (Zvifananidzo Zvekuwedzera 6a,b).
Mushure mekuongorora kwekutanga, takaita kuenzanisa mamiriro mana ekurima (Mufananidzo 4a): Ctrl: zvikamu zvemoyo zvakarimwa mutsika yedu yakatsanangurwa kare tichishandisa nzira yedu yakagadziriswa; 20.21 TD: T3 uye Ctrl s Added Dex neChitatu; MC: zvikamu zvemoyo zvakarimwa muCTCM tichishandisa nzira yedu yakagadziriswa kare; uye MT: CTCM ine T3 uye Dex yakawedzerwa kune nzira. Mushure memazuva gumi nemaviri ekurima, kugona kweMS neMT tishu kwakaramba kwakafanana nekwemunyama itsva dzakaongororwa neMTT assay (Mufananidzo 4b). Zvinofadza kuti kuwedzerwa kweT3 neDex ku transwell cultures (TD) hakuna kukonzera kuvandudzika kukuru mukurarama zvichienzaniswa nemamiriro eCtrl, zvichiratidza basa rakakosha rekukurudzira kwemakemikari mukuchengetedza kugona kwezvikamu zvemoyo.
Dhayagiramu yekuedza inoratidza mamiriro mana ekurima anoshandiswa kuongorora mhedzisiro yekukurudzira kwemakemikari uye kuwedzerwa kweT3/Dex panzvimbo yepakati kwemazuva gumi nemaviri. b Girafu yebhawa inoratidza kuyera kwekushanda kwemazuva gumi nemaviri mushure mekurimwa mumamiriro ese mana ekurimwa (Ctrl, TD, MC, uye MT) zvichienzaniswa nezvidimbu zvemoyo zvitsva (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD uye D12 MT), zvidimbu gumi nezviviri (D12 MC) kubva munguruve dzakasiyana, bvunzo yeANOVA inoitwa nenzira imwe chete; ####p < 0.0001, ###p < 0.001 zvichienzaniswa naD0 uye **p < 0.01 zvichienzaniswa naD12 Ctrl). b Girafu yebhawa inoratidza kuyera kwekushanda kwemazuva gumi nemaviri mushure mekurimwa mumamiriro ese mana ekurimwa (Ctrl, TD, MC, uye MT) zvichienzaniswa nezvidimbu zvemoyo zvitsva (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD uye D12 MT), zvidimbu gumi nezviviri (D12 MC) kubva munguruve dzakasiyana, bvunzo yeANOVA inoitwa nenzira imwe chete; ####p < 0.0001, ###p < 0.001 zvichienzaniswa naD0 uye **p < 0.01 zvichienzaniswa naD12 ctrl). b Гистограмма показывает количественную оценку жизнеспособности через 12 дней после культивирования во всех 4 условиях культивирования во всех 4 условиях культивирования сравнению со свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD uye D12 MT), 12 (D12 MC) срезов/группу от разнстрой свинство ANOVA; ####p <0,0001, ###p < 0,001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). b Girafu yebhawa inoratidza huwandu hwekushanda kwemazuva gumi nemaviri mushure mekurimwa mumamiriro ese mana ekurimwa (kutonga, TD, MC, uye MT) zvichienzaniswa nezvikamu zvemoyo mutsva (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, uye D12 MT), zvikamu gumi nezviviri (D12 MC) kubva kunguruve dzakasiyana, bvunzo yeANOVA yenzira imwe chete; ####p < 0.0001, ###p < 0.001 vs. D0 uye **p < 0.01 by zvichienzaniswa neD12 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,D12 TD) MT)，來自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；####p < 0.0001,###p < 0.001 与D0 相比，*0p与D12控制）.b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия культивирования (контроль, TD, MC ne MT) по сравнению свежими срезами сердца (D0) (n = 15,D12), D0 MT), от разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA; b Histogram inoratidza mamiriro ese mana ekurera (kutonga, TD, MC uye MT) zvichienzaniswa nezvikamu zvemoyo mutsva (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD uye D12 MT), kubva kunguruve dzakasiyana 12 (D12 MC) zvikamu/boka, bvunzo yeANOVA yenzira imwe chete; ####p<0.0001, ###p<0.001 vs. D0, **p<0.01 vs. kutonga D12). c Bar graph inoratidza kuyera kweglucose flux mazuva gumi nemaviri mushure mekukura muzvirwere zvese zvina (Ctrl, TD, MC, uye MT) zvichienzaniswa nezvidimbu zvemoyo zvitsva (D0) (n = zvidimbu zvitanhatu/boka kubva kunguruve dzakasiyana, bvunzo yeANOVA inoitwa nenzira imwe chete; ###p < 0.001, zvichienzaniswa naD0 uye ***p < 0.001 zvichienzaniswa naD12 Ctrl). c Bar graph inoratidza kuyera kweglucose flux mazuva gumi nemaviri mushure mekukura muzvirwere zvese zvina (Ctrl, TD, MC, uye MT) zvichienzaniswa nezvidimbu zvemoyo zvitsva (D0) (n = zvidimbu zvitanhatu/boka kubva kunguruve dzakasiyana, bvunzo yeANOVA inoitwa nenzira imwe chete; ###p < 0.001, zvichienzaniswa naD0 uye ***p < 0.001 zvichienzaniswa naD12 Ctrl). c Гистограмма показывает количественную оценку потока глюкозы через 12 дней после культивирования во всех 4 условиях культивирования, което сравнению со свежими срезами сердца (D0) (n = 6 срезов/группу от разных, односторонний Выполняется тест ANOVA; по сравнению с D12 Ctrl). c Histogram inoratidza kuyera kweglucose flux mazuva gumi nemaviri mushure mekurimwa pasi pemamiriro ese mana ekurimwa (kutonga, TD, MC uye MT) zvichienzaniswa nezvikamu zvemoyo mutsva (D0) (n = zvikamu zvitanhatu/boka kubva kunguruve dzakasiyana, bvunzo yeANOVA yakaitwa nenzira imwe chete; ###p < 0.001 zvichienzaniswa neD0 uye ***p < 0.001 zvichienzaniswa neD12 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12天的葡萄糖通量定量（n = 6 片/组，來自不同猪，单向执行ANOVA 测试；###p <0.001，与D0 相0p1 20 Shona*.相比）. C 条形图 显示 所有 4 种 条件 （ctrl 、 td 、 mc 、 mc 和 mt) 12 天 的 通量 定量 （n = 6 片/组 ， 來自 猪 , , , , , , , , 猪 猪单向执行ANOVA 测试；#0#01 <0.相比,***p < 0.001 kuD12 Ctrl 相比）. c Гистограмма, показывающая количественную оценку потока глюкозы через 12 дней после культивирования для всех 4 сравнению со свежими срезами сердца (D0) (n = 6 срезов/группа, от разных свиней, односторонний Были проведены тесты ANOVA; ###0p < 0,0 0,001 по сравнению с D12 (контроль). c Histogram inoratidza kuyera kweglucose flux pamazuva gumi nemaviri mushure mekurimwa kwezvirwere zvese zvina zvekurimwa (control, TD, MC, uye MT) zvichienzaniswa nezvikamu zvemoyo mutsva (D0) (n = zvikamu zvitanhatu/boka, kubva kunguruve dzakasiyana, unilateral. Pakaitwa bvunzo dzeANOVA, ###p < 0.001 zvichienzaniswa naD0, ***p < 0.001 zvichienzaniswa naD12 (control).d Mapuratifomu ekuongorora kusvuta ematishu matsva (ebhuruu), zuva rechi12 MC (girini), uye zuva rechi12 MT (tsvuku) pamapoinzi gumi echikamu chematishu edunhu (n = zvidimbu zvina/boka, bvunzo yeANOVA yenzira imwe chete; pakanga pasina musiyano wakakosha pakati pemapoka). e Plot yekuputika kwegomo inoratidza majini akaratidzwa zvakasiyana muzvikamu zvemoyo mutsva (D0) zvichienzaniswa nezvikamu zvemoyo zvakakuriswa pasi pemamiriro ezvinhu asingachinji (Ctrl) kana pasi pemamiriro ezvinhu eMT (MT) kwemazuva gumi kusvika gumi nemaviri. f Mepu yekupisa yemajini esarcomere ezvikamu zvemoyo zvakakuriswa pasi pemamiriro ega ega emamiriro ekurima. Mitsetse yekukanganisa inomiririra avhareji ± kutsauka kwakajairika.
Kuvimba nekuchinja kwemetabolism kubva pa fatty acid oxidation kuenda ku glycolysis chiratidzo chekubviswa kwe cardiomyocyte. Cardiomyocytes dzisina kukura dzinonyanya kushandisa glucose pakugadzira ATP uye dzine hypoplastic mitochondria ine cristae shoma5,32. Kuongororwa kwekushandiswa kweglucose kwakaratidza kuti pasi pemamiriro eMC neMT, kushandiswa kweglucose kwakafanana nekwemumatishu ezuva 0 (Mufananidzo 4c). Zvisinei, Ctrl samples yakaratidza kuwedzera kukuru mukushandiswa kweglucose zvichienzaniswa nematishu matsva. Izvi zvinoratidza kuti kusanganiswa kweCTCM neT3/Dex kunowedzera simba rematishu uye kunochengetedza metabolic phenotype yezvikamu zvemoyo zvemazuva gumi nemaviri zvakamera. Pamusoro pezvo, ongororo yestrain yakaratidza kuti strain levels yakaramba yakafanana neyemumatishu matsva emwoyo kwemazuva gumi nemaviri pasi pemamiriro eMT neMS (Mufananidzo 4d).
Kuti tiongorore kukanganisa kweCTCM neT3/Dex pamamiriro epasi rose ekunyora kwenyama yemwoyo, takaita RNAseq pazvidimbu zvemwoyo kubva mumamiriro ese mana akasiyana etsika (Supplementary Data 1). Zvinofadza kuti zvikamu zveMT zvakaratidza kufanana kwakanyanya kwekunyora kwenyama yemwoyo mitsva, ne16 chete yakaratidzwa zvakasiyana kubva mumajini 13,642. Zvisinei, sezvatakaratidza kare, zvidimbu zveCtrl zvakaratidza majini 1229 akaratidzwa zvakasiyana mushure memazuva 10-12 mukurima (Mufananidzo 4e). Aya data akasimbiswa neqRT-PCR yemajini emwoyo nefibroblast (Mufananidzo 7a-c). Zvinofadza kuti zvikamu zveCtrl zvakaratidza kudzikira kwemajini emwoyo nemasero uye kushanda kwemapurogiramu emajini ekuzvimba. Aya data anoratidza kuti dedifferentiation, iyo inowanzoitika mushure mekurima kwenguva refu, inodzikira zvachose pasi pemamiriro eMT (Mufananidzo 8a,b). Kunyatsoongorora majini e sarcomere kwakaratidza kuti majini anoumba sarcomere (Mufananidzo 4f) uye ion channel (Mufananidzo 9) chete ndiwo anochengetedzwa, achiadzivirira kubva mukudzvinyirirwa pasi pemamiriro e Ctrl, TD, uye MC. Aya data anoratidza kuti nekubatanidzwa kwe mechanical uye humoral stimulation (T3/Dex), heart slice transcriptome inogona kuramba yakafanana ne heart slices itsva mushure memazuva gumi nemaviri ekurera.
Zvakawanikwa izvi zvinotsigirwa nekuti kusimba kwechimiro chemwoyo muzvikamu zvemoyo kunochengetedzwa zvakanaka pasi pemamiriro eMT kwemazuva gumi nemaviri, sezvakaratidzwa ne connexin 43 isina kukuvadzwa uye iri munzvimbo (Mufananidzo 5a). Pamusoro pezvo, fibrosis muzvikamu zvemoyo pasi pemamiriro eMT yakaderera zvakanyanya zvichienzaniswa ne Ctrl uye yakafanana nezvikamu zvemoyo mutsva (Mufananidzo 5b). Data iri rinoratidza kuti kusanganiswa kwekukurudzira kwemuchina uye kurapwa kweT3/Dex zvinochengetedza chimiro chemwoyo muzvikamu zvemoyo mutsika.
Mifananidzo inoratidza immunofluorescence yetroponin-T (girini), connexin 43 (tsvuku), uye DAPI (bhuruu) muzvikamu zvemwoyo zvitsva (D0) kana kurimwa kwemazuva gumi nemaviri mumamiriro ese mana ekukura kwechikamu chemwoyo (scale bar = 100 µm). Kuyerwa kwehungwaru hwekugadzira kwehunhu hwemoyo (n = 7 (D0 naD12 Ctrl), 5 (D12 TD, D12 MC naD12 MT) zvidimbu/boka kubva kunguruve dzakasiyana, bvunzo yeANOVA yenzira imwe chete inoitwa; ####p < 0.0001 zvichienzaniswa naD0 na *p < 0.05, kana ****p < 0.0001 zvichienzaniswa naD12 Ctrl). Kuyerwa kwehungwaru hwekugadzira kwehunhu hwemoyo (n = 7 (D0 naD12 Ctrl), 5 (D12 TD, D12 MC naD12 MT) zvidimbu/boka kubva kunguruve dzakasiyana, bvunzo yeANOVA yenzira imwe chete inoitwa; #### p < 0.0001 zvichienzaniswa naD0 na *p < 0.05, kana ****p < 0.0001 zvichienzaniswa naD12 Ctrl). Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта (n = 7 (D0 uye D12 Ctrl), 5 (D12 TD, D12 MT/ D12 MT/ Dпре) от разных свиней, проведен однофакторный тест ANOVA; Kuyera kusimba kwechimiro chemuviri wemoyo uchishandisa njere dzekugadzira (n = 7 (D0 naD12 Ctrl), 5 (D12 TD, D12 MC naD12 MT) zvikamu/boka kubva kunguruve dzakasiyana, bvunzo yeANOVA yakaitwa nenzira imwe chete; #### p < 0.0001 zvichienzaniswa naD0 na *p < 0.05 kana ****p < 0.0001 zvichienzaniswa naD12 Ctrl).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）进行人工智能量化，进行单向ANOVA 测试;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 mt）智能量 化 进行 单向 单向 单向 测试 ;相比）.Kuyera kusimba kwechimiro chenyama yemwoyo uchishandisa njere dzekugadzira munguruve dzakasiyana (n = 7 (D0 naD12 Ctrl), 5 (D12 TD, D12 MC naD12 MT) zvikamu/boka) nebvunzo yeANOVA yenzira imwe chete;#### p < 0,0001 по сравнению с D0 и *p < 0,05 или ****p < 0,0001 по сравнению с D12 Ctrl). #### p < 0.0001 zvichienzaniswa naD0 uye *p < 0.05 kana ****p < 0.0001 zvichienzaniswa naD12 Ctrl). b Mifananidzo inomiririra uye kuyera kwezvidimbu zvemoyo zvakapendwa neMasson's trichrome stain (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, uye D12 MC), zvidimbu 9 (D12 MT) kubva munguruve dzakasiyana, bvunzo yeANOVA inoitwa nenzira imwe chete; ####p < 0.0001 zvichienzaniswa naD0 uye ***p < 0.001, kana ****p < 0.0001 zvichienzaniswa naD12 Ctrl). b Mifananidzo inomiririra uye kuyera kwezvidimbu zvemoyo zvakapendwa neMasson's trichrome stain (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, uye D12 MC), zvidimbu 9 (D12 MT) kubva munguruve dzakasiyana, bvunzo yeANOVA inoitwa nenzira imwe chete; ####p < 0.0001 zvichienzaniswa naD0 uye ***p < 0.001, kana ****p < 0.0001 zvichienzaniswa naD12 Ctrl). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Массона (масштабная 10) = 10 = 10 (D0, D12 Ctrl, D12 TD uye D12 MC), 9 (D12 MT) срезов/группу от разных свиней, выполняется односторонний тест ANOVA < ####pнию <0,0 *** 0,001 uye ****p < 0,0001 по сравнению с D12 Ctrl). b Mifananidzo inomiririra uye kuyera zvikamu zvemoyo zvakapendwa neMasson's trichrome stain (scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD uye D12 MC), zvikamu 9 (D12 MT)/boka kubva kunguruve dzakasiyana, zvakaitwa nenzira imwe chete ANOVA; ####p < 0.0001 vs. D0 uye ***p < 0.001 kana ****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 = 11(D2 TD), D和MC. 0.0001 kuD12 Ctrl 相比）. 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 = 10 （d0 、 d12 ctrl 、 d12 td 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 切片 切片/组，进行单因素方差分析; 0.0001 D12 Ctrl 相比）. b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромом Массона (масштабная линейка = 500, Ctrl = 500, D1 = 500, D12 TD uye D12 MC), 9 (D12 MT) срезов от разных свиней / группы, один- способ ANOVA; D12 Ctrl). b Mifananidzo inomiririra uye kuyera zvikamu zvemoyo zvakapendwa neMasson's trichrome (scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD uye D12 MC), zvikamu 9 (D12 MT) kubva kunguruve/boka rakasiyana, nzira imwe chete yeANOVA; ####p < 0.0001 zvichienzaniswa neD0, ***p < 0.001 kana ****p < 0.0001 zvichienzaniswa neD12 Ctrl).Mitsetse yekukanganisa inomiririra avhareji ± kutsauka kwakajairika.
Pakupedzisira, kugona kweCTCM kutevedzera hypertrophy yemwoyo kwakaongororwa nekuwedzera kutambanudza kwenyama yemwoyo. MuCTCM, kumanikidzwa kwemhepo kwakanyanya kwakawedzera kubva pa80 mmHg kusvika pa80 mmHg. Art. (kutambanudza kwakajairika) kusvika pa140 mmHg Art. (Mufananidzo 6a). Izvi zvinoenderana nekuwedzera kwekutambanudza kwe32% (Mufananidzo 6b), uko kwakamboratidzwa sechikamu chinoenderana chekutambanudza chinodiwa kuti zvikamu zvemoyo zviwane hurefu hwesarcomere hwakafanana nehunoonekwa muhypertrophy. Kutambanudza nekumhanya kwenyama yemwoyo panguva yekudzikira nekureruka kwakaramba kwakafanana mukati memazuva matanhatu ekukudziridza (Mufananidzo 6c). Nyama yemwoyo kubva mumamiriro eMT yakaiswa kutambanudza kwakajairika (MT (Normal)) kana mamiriro ekutambanudza zvakanyanya (MT (OS)) kwemazuva matanhatu. Mushure memazuva mana ekukudziridza, hypertrophic biomarker NT-ProBNP yakakwira zvakanyanya munzvimbo iri pasi pemamiriro eMT (OS) zvichienzaniswa nemamiriro eMT (normal) (Mufananidzo 7a). Pamusoro pezvo, mushure memazuva matanhatu ekurera, saizi yemasero muMT (OS) (Mufananidzo 7b) yakawedzera zvakanyanya zvichienzaniswa nezvikamu zvemwoyo weMT (zvakajairika). Pamusoro pezvo, NFATC4 nuclear translocation yakawedzera zvakanyanya mumatishu akatambanudzwa zvakanyanya (Mufananidzo 7c). Mhedzisiro iyi inoratidza kufambira mberi kwekuvandudzwa kwepathological mushure mehyperdistension uye inotsigira pfungwa yekuti mudziyo weCTCM unogona kushandiswa sepuratifomu yekudzidza stretch-induced cardiac hypertrophy signaling.
Zviratidzo zvinoratidza kumanikidzwa kwemukamuri yemhepo, kumanikidzwa kwemukamuri yemvura, uye kuyerwa kwekufamba kwenyama zvinosimbisa kuti kumanikidzwa kwemukamuri kunochinja kumanikidzwa kwemukamuri yemvura, zvichikonzera kufamba kwakafanana kwechidimbu chenyama. b Zvinomiririra chikamu chekutambanudza uye mwero wekutambanudza wezvikamu zvenyama zvinowanzotambanudzwa (orenji) uye zvakatambanudzwa zvakanyanya (bhuruu). c Girafu yebha inoratidza nguva yekutenderera (n = zvidimbu 19 paboka, kubva kunguruve dzakasiyana), nguva yekukotama (n = zvidimbu 18-19 paboka, kubva kunguruve dzakasiyana), nguva yekuzorora (n = zvidimbu 19 paboka, kubva kunguruve dzakasiyana) ), amplitude yekufamba kwenyama (n = zvidimbu 14/boka, kubva kunguruve dzakasiyana), peak systolic velocity (n = zvidimbu 14/boka, kubva kunguruve dzakasiyana) uye peak relaxation rate (n = 14 (D0), 15 (D6) ) zvikamu/mapoka) kubva kunguruve dzakasiyana), bvunzo yeT-test yeMudzidzi ine miswe miviri haina kuratidza mutsauko wakakosha mune chero parameter, zvichiratidza kuti ma parameter aya akaramba akafanana mukati memazuva matanhatu ekukudziridza ne overvoltage. Error bars inomiririra avhareji ± standard deviation.
Kuyera huwandu hweNT-ProBNP mu culture media kubva kuzvidimbu zvemoyo zvakakuriswa pasi peMT normal stretch (Norm) kana overstretching (OS) mamiriro (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, uye D4 MTOS) zvidimbu/boka kubva kunguruve dzakasiyana, Two-way ANOVA inoitwa; **p < 0.01 zvichienzaniswa nekutambanudza kwakajairika). Kuyera huwandu hweNT-ProBNP muchikamu che culture media kubva kuzvikamu zvemoyo zvakakuriswa pasi pemamiriro ekutambanudza kwakajairika (Norm) kana kutambanudza zvakanyanya (OS) (n ​​= 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, uye D4 MTOS) zvidimbu/boka kubva kunguruve dzakasiyana, Two-way ANOVA inoitwa; **p < 0.01 zvichienzaniswa nekutambanudza kwakajairika).Huwandu hwehuwandu hweNT-ProBNP muchikamu chekurima kubva pazvidimbu zvemoyo zvakakuriswa pasi pemamiriro ezvinhu akajairwa ekutambanudza MT (norm) kana kutambanudza zvakanyanya (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, uye D4).MTOS) zvidimbu /boka kubva kunguruve dzakasiyana, ongororo yekuchinja kwemhando mbiri inoitwa;**p < 0,01 по сравнению с нормальным растяжением). **p < 0.01 zvichienzaniswa nekutambanudza kwakajairika). 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 浓麦的条彈n = 2 MTNorm)、 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1 . a Quantification ye NT-ProBNP kusungirirwa muzvikamu zvemoyo zvakasimudzwa pasi peMT yakajairika kutambanudza (Nyevedzo) kana kuwandisa (OS) mamiriro (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm neD4 MTOS) kubva kwakasiyana 猪的切片/组，可以双向neyakajairwa kutambanudza, p <0.01).histogram Kuyera huwandu hweNT-ProBNP muzvidimbu zvemoyo zvakarimwa pasi pemamiriro ezvinhu akajairwa ekutambanudza MT (norm) kana kutambanudza zvakanyanya (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) uye D4 MTOS) zvidimbu/boka kubva kunguruve dzakasiyana, kuongorora musiyano wenzira mbiri;**p < 0,01 по сравнению с нормальным растяжением). **p < 0.01 zvichienzaniswa nekutambanudza kwakajairika). b Mifananidzo inomiririra yezvidimbu zvemoyo zvakapendwa netroponin-T neWGA (kuruboshwe) uye chiyero chehukuru hwemasero (kurudyi) (n = 330 (D6 MTOS), 369 (D6 MTNorm) masero/boka kubva kuzvidimbu gumi zvakasiyana kubva kunguruve dzakasiyana, Two-tailed Student t-test inoitwa; ****p < 0.0001 zvichienzaniswa nekutambanudza kwakajairika). b Mifananidzo inomiririra yezvidimbu zvemoyo zvakapendwa netroponin-T neWGA (kuruboshwe) uye chiyero chehukuru hwemasero (kurudyi) (n = 330 (D6 MTOS), 369 (D6 MTNorm) masero/boka kubva kuzvidimbu gumi zvakasiyana kubva kunguruve dzakasiyana, T-test yeMudzidzi ine miswe miviri inoitwa; ****p < 0.0001 zvichienzaniswa nekutambanudza kwakajairika). b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественного определения размера клеток (с) = 6 369 (D6 MTNorm) клеток/группу из 10 разных срезов от разных свиней, два- проводится хвостовой t-критерий Стьюден ****pыдента ****pымном ****pымнта ****pымнта растяжением). b Mifananidzo inomiririra yezvikamu zvemoyo zvakapendwa netroponin-T neAZP (kuruboshwe) uye chiyero chehukuru hwemasero (kurudyi) (n = 330 (D6 MTOS), 369 (D6 MTNorm) masero/boka kubva muzvikamu gumi zvakasiyana kubva kunguruve dzakasiyana, bvunzo yeT-yeMudzidzi ine miswe miviri yakaitwa; ****p < 0.0001 zvichienzaniswa nekuoma kwakajairika). b 用肌钙蛋白-T 和WGA（左）和细胞大小量化（右）染色的心脏切片的代表性图像（右）染色的心脏切片的代表性图像（n =D60) MTOS），來自不同猪的10 个不同切片的369（D6 MTNorm）细胞/组，两进行有尾学生t 检验；与正卸0.0001. b Mifananidzo inomiririra yezvidimbu zvemoyo zvakapendwa ne calcarein-T ne WGA (kuruboshwe) uye saizi yesero (kurudyi) (n = 330 (D6 MTOS), 369 kubva kuzvidimbu gumi zvakasiyana (D6 MTNorm)) Masero/组，两方法有尾学生t test； zvichienzaniswa nekutambanudza kwakajairika，****p < 0.0001). b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественная оценка размера клеток (S69 = MT63) (D6 MTNorm) из 10 различных срезов от разных свиней Клетки/группа, двусторонние критерий Стьюдента ****p < 0,0001 по сравнильмы . b Mifananidzo inomiririra yezvikamu zvemoyo zvakapendwa netroponin-T neAZP (kuruboshwe) uye kuyera saizi yesero (kurudyi) (n = 330 (D6 MTOS), 369 (D6 MTNorm) kubva muzvikamu gumi zvakasiyana kubva kunguruve dzakasiyana) Masero/boka, chiyero chine miswe miviri T yemudzidzi; ****p < 0.0001 zvichienzaniswa nekuoma kwakajairika). c Mifananidzo inomiririra yezuva 0 nerezuva rechi6 MTOS heart slices dzakaiswa immunolabeled ye troponin-T ne NFATC4 uye chiyero chekutamiswa kweNFATC4 kuenda ku nuclei dzeCMs (n = 4 (D0), 3 (D6 MTOS) slices/group kubva kunguruve dzakasiyana, Two-tailed Student t-test inoitwa; *p < 0.05). c Mifananidzo inomiririra yezuva 0 nerezuva rechi6 MTOS heart slices dzakaiswa immunolabeled ye troponin-T ne NFATC4 uye chiyero chekutamiswa kweNFATC4 kuenda ku nuclei dzeCMs (n = 4 (D0), 3 (D6 MTOS) slices/group kubva kunguruve dzakasiyana, Two-tailed Student t-test inoitwa; *p < 0.05). c Репрезентативные изображения для срезов сердца 0 и 6 дней MTOS, иммуномеченых для тропонина-Т и NFATC4, и количественная оценская кавернозных клеток (n = 4 (D0), 3 (D6 MTOS) срезов/группу от разных свиней , выполняется двусторонний t-критерий Стьюдента, *5. c Mifananidzo inomiririra zvikamu zvemoyo pamazuva 0 ne6 eMTOS, yakaisirwa immunolabeled yetroponin-T neNFATC4, uye kuyera kweNFATC4 translocation munucleus yemasero emumapako (n = 4 (D0), 3 (D6 MTOS) zvidimbu/boka kubva kunguruve dzakasiyana) yakaita bvunzo yeT-yeMudzidzi ine miswe miviri; *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，以及來自肉TCCM4细胞核的量化（n = 4 (D0), 3 (D6 MTOS) 切片/组, 进行双尾学生t 检验；*p <0.05). c Inomiririra mifananidzo ye calcanin-T uye NFATC4 immunolabeling 第0天和第6天MTOS moyo zvimedu, uye NFATC4 kubva akasiyana NFATC4 易位至CM cell nucleus的quantity化 (n = 4 (D0), 3 (D6 MTOS) / 切礼时间双尾学生et 电影；*p <0.05). c Репрезентативные изображения срезов сердца MTOS на 0 и 6 день для иммуномаркировки тропонином-Т и NFATC4 и количественная оценская оценка от разных свиней (n = 4 (D0), 3 (D6 MTOS) срез/группа, два- хвостатый t-критерий Стьюдента; *p <0,05). c Mifananidzo inomiririra zvidimbu zvemwoyo zveMTOS pazuva re0 nerechi6 zve troponin-T neNFATC4 immunolabeling uye quantification yeNFATC4 translocation mu nucleus yeCM kubva kunguruve dzakasiyana (n = 4 (D0), 3 (D6 MTOS) zvidimbu/boka, t-criterion ine miswe miviri. Vadzidzi; *p < 0.05).Mitsetse yekukanganisa inomiririra avhareji ± kutsauka kwakajairwa.
Ongororo yezvemwoyo inoshandura mafambiro emwoyo inoda mamodheru emasero anoburitsa zvakanaka mamiriro emwoyo. Muchidzidzo ichi, mudziyo weCTCM wakagadzirwa uye wakaratidzwa unogona kukurudzira zvikamu zvitete zvemoyo. Sisitimu yeCTCM inosanganisira physiologically synchronized electromechanical stimulation uye T3 neDex fluid enrichment. Zvikamu zvemoyo wenguruve pazvakaratidzwa kune izvi, kushanda kwazvo, chimiro chazvo, mashandiro emetabolism, uye kunyorwa kwetranscriptional kwakaramba kwakafanana nekwenyama yemoyo mutsva mushure memazuva gumi nemaviri ekurera. Pamusoro pezvo, kutambanudza zvakanyanya kwenyama yemwoyo kunogona kukonzera hypertrophy yemoyo inokonzerwa nehyperextension. Zvose izvi, mhedzisiro iyi inotsigira basa rakakosha remamiriro emuviri mukuchengetedza phenotype yemwoyo yakajairika uye inopa nzvimbo yekuongororwa kwemishonga.
Zvinhu zvakawanda zvinobatsira pakugadzira nzvimbo yakakwana yekushanda uye kupona kwe cardiomyocytes. Zvinhu zviri pachena pane izvi zvine chekuita ne (1) kudyidzana pakati pemasero, (2) electromechanical stimulation, (3) humoral factors, uye (4) metabolic substrates. Kudyidzana kwe physiological cell-to-cell kunoda network dzakaoma dzemhando nhatu dzemasero akawanda dzinotsigirwa ne extracellular matrix. Kudyidzana kwakadai kwakaomarara kwemasero kwakaoma kuvakazve in vitro kuburikidza ne co-culture yemarudzi emasero ega ega, asi zvinogona kuwanikwa zviri nyore uchishandisa organotypic hunhu hwezvikamu zvemoyo.
Kutambanudza kwesimba uye kukurudzira kwemagetsi kwemwoyo yemwoyo zvakakosha pakuchengetedza phenotype yemwoyo33,34,35. Kunyange hazvo kukurudzira kwesimba kwave kuchishandiswa zvakanyanya pakugadzirisa uye kukura kwemoyo nehuropi hwe ... Kunyangwe izvi zvekukurudzira kwemuviri zvine mativi matatu chete zvisingakwani kuchengetedza hutano hwemuviri, musanganiswa wekukurudzira kwemuviri nekwemuviri uchishandisa T3/Dex unodiwa kuti muviri ugare zvakanaka, ushande zvakanaka, uye hunhu hwakanaka.
Zvinhu zvinosetsa zvinoita basa rakakosha mukugadzirisa chimiro chemwoyo wemunhu mukuru. Izvi zvakaratidzwa muzvidzidzo zveHiPS-CM umo T3 neDex zvakawedzerwa kune culture media kuti kukurumidze kukura kwemasero. T3 inogona kukanganisa kutakurwa kweamino acids, shuga uye calcium kuburikidza ne membranes dzemasero36. Pamusoro pezvo, T3 inokurudzira MHC-α expression uye kudzikiswa kweMHC-β, zvichikurudzira kuumbwa kwe fast twitch myofibrils mu cardiomyocytes dzakakura zvichienzaniswa ne slow twitch myofibrils mu fetal CM. Kushaikwa kweT3 muvarwere vane hypothyroidism kunoguma nekurasikirwa kwe myofibrillar bands uye kudzikira kwekukura kwetoni37. Dex inoshanda pama glucocorticoid receptors uye yakaratidzwa kuti inowedzera myocardial contractility mumoyo yakaparadzaniswa yakapera;38 kuvandudzika uku kunofungidzirwa kuti kune chekuita nemhedzisiro pa calcium deposit-driven entry (SOCE) 39,40. Pamusoro pezvo, Dex inosunga kuma receptors ayo, zvichikonzera mhinduro yakakura yemukati memasero inodzvinyirira mashandiro ekudzivirira muviri uye kuzvimba30.
Zvakabuda muongororo yedu zvinoratidza kuti physical mechanical stimulation (MS) yakavandudza mashandiro emuviri wese zvichienzaniswa neCtrl, asi yakakundikana kuchengetedza kushanda zvakanaka, structural integrity, uye cardiac expression kwemazuva gumi nemaviri muculture. Zvichienzaniswa neCtrl, kuwedzerwa kweT3 neDex kuCTCM (MT) cultures kwakavandudza kushanda zvakanaka uye kwakachengetedza ma profiles akafanana e transcription, structural integrity, uye metabolic activity ne fresh heart tissue kwemazuva gumi nemaviri. Pamusoro pezvo, nekudzora mwero wekutambanudza kwe tissue, hyperextension-induced cardiac hypertrophy model yakagadzirwa ichishandisa STCM, ichiratidza kushanduka-shanduka kweSTCM system. Zvinofanira kucherechedzwa kuti kunyangwe cardiac remodeling uye fibrosis zvinowanzo sanganisira nhengo dzisina kusimba dzine masero anotenderera anogona kupa ma cytokines akakodzera pamwe ne phagocytosis nezvimwe zvinhu zvinogadzirisa, zvikamu zvemoyo zvinogona kutevedzera fibrotic process mukupindura kushushikana nekukuvara. Izvi zvakamboongororwa mu cardiac slice model iyi. Zvinofanira kucherechedzwa kuti CTCM parameters dzinogona kugadziriswa nekuchinja pressure/electrical amplitude uye frequency kuti zvitevedzere mamiriro akawanda akadai se tachycardia, bradycardia, uye mechanical circulatory support (mechanical unloaded heart). Izvi zvinoita kuti sisitimu iyi ive nzira iri pakati nepakati yekuyedzwa kwemishonga. Kugona kweCTCM kuratidza hypertrophy yemwoyo inokonzerwa nekushanda nesimba kwakanyanya kunovhura nzira yekuedza sisitimu iyi kuti iwane kurapwa kwakagadzirirwa munhu. Mukupedzisa, chidzidzo ichi chinoratidza kuti kutambanudza kwemuviri uye kukurudzira humoral zvakakosha pakuchengetedza tsika yezvikamu zvenyama yemwoyo.
Kunyangwe data rakapihwa pano richiratidza kuti CTCM inzvimbo inovimbisa zvikuru yekutevedzera myocardium isina kusimba, nzira iyi yekukudziridza ine zvimwe zvipingamupinyi. Muganho mukuru weCTCM culture ndewekuti unoisa kushushikana kwesimba remagetsi kunoenderera mberi pazvidimbu, izvo zvinodzivisa kugona kutarisa kurova kwemoyo panguva yega yega kutenderera. Pamusoro pezvo, nekuda kwehukuru hudiki hwezvikamu zvemwoyo (7 mm), kugona kuongorora mashandiro esystolic kunze kwemasystem ekusimudzira uchishandisa masensa esimba echinyakare kwakaderera. Muchinyorwa chazvino, tinokunda zvishoma muganho uyu nekuongorora voltage ye optical sechiratidzo chebasa re contractile. Zvisinei, muganho uyu uchada basa rakawanda uye unogona kugadziriswa mune ramangwana nekuisa nzira dzekutarisa mashandiro ezvikamu zvemoyo mukusimudzira, senge optical mapping uchishandisa calcium uye voltage-sensitive dyes. Chimwe chipingamupinyi cheCTCM ndechekuti modhi inoshanda haitungamirire kushushikana kwemuviri (preload uye afterload). MuCTCM, kumanikidzwa kwakakonzerwa nenzira dzakasiyana kuti kubereke 25% physiological stretch mu diastole (full stretch) uye systole (hurefu hwekukombana panguva yekukurudzira kwemagetsi) mumatishu makuru kwazvo. Muganho uyu unofanira kubviswa mune ramangwana reCTCM nekudzvanya kwakakwana panyama yemwoyo kubva kumativi ese uye nekushandisa hukama chaihwo hwekudzvanya nehuwandu hunoitika mumakamuri emwoyo.
Kugadziriswa kwakaitwa zvakanyanya kwakanyorwa muchinyorwa chino kwakaganhurirwa pakutevedzera zviratidzo zve hypertrophic hyperstretch. Saka, modhi iyi inogona kubatsira mukudzidza zviratidzo zve hypertrophic zvinotambanudzwa pasina chikonzero che humoral kana neural factors (izvo zvisiri muhurongwa uhwu). Zvimwe zvidzidzo zvinodiwa kuti kuwedzere kuwanda kweCTCM, semuenzaniso, kushandira pamwe chete nemasero ekudzivirira muviri, circulating plasma humoral factors, uye innervation kana kubatanidzwa pamwe chete nemasero e neuronal zvichavandudza mikana yekutevedzera chirwere neCTCM.
Nguruve gumi nenhatu dzakashandiswa muchidzidzo ichi. Maitiro ese emhuka akaitwa zvichienderana nemirairo yesangano uye akatenderwa neUniversity of Louisville Institutional Animal Care and Use Committee. Aortic arch yakasungwa uye moyo wakazorwa ne 1 L ye sterile cardioplegia (110 mM NaCl, 1.2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 U/mL heparin, pH kusvika 7.4); Mwoyo yacho yaichengetwa mu cardioplegic solution inotonhora kusvika yaendeswa ku lab iri pachando iyo inowanzova pasi pemaminitsi gumi. Mwoyo yacho yaichengetwa mu cardioplegic solution inotonhora kusvika yaendeswa ku lab iri pachando iyo inowanzova pasi pemaminitsi gumi. Сердца хранили в ледяном кардиоплегическом растворе до транспортировки в лабораторию на льду, что обычно занимает <10 мин. Mwoyo yakachengetwa mu cardioplegic solution inotonhora zvikuru kusvika yaendeswa ku laboratory iri muchando, iyo inowanzotora pasi pemaminitsi gumi.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. Chengeta mwoyo iri pachando kusvika yaendeswa kurabhoritari iri pachando, kazhinji pasi pemaminitsi gumi.
Mudziyo weCTCM wakagadzirwa muSolidWorks computer-aid design (CAD) software. Makamuri ekurera, madivider uye maair chambers akagadzirwa neCNC clear acrylic plastic. Ring yekudzosera ine dhayamita ye7mm yakagadzirwa nehigh density polyethylene (HDPE) pakati uye ine o-ring groove inokwana silicone o-ring inoshandiswa kuvhara media iri pasi. Kamudzira kesilica katete kanoparadzanisa kamudzira kekusikira kubva paplate yekuparadzanisa. Kamudzira kesilica kakachekwa nelaser kubva papepa resilica rine gobvu re0.02″ uye kane kuomarara kwe35A. Magasket esilica epasi nepamusoro akachekwa nelaser kubva papepa resilica rine gobvu re1/16″ uye akane kuomarara kwe50A. Zvikuruu zvesimbi isina tsvina zve316L uye mawing nuts zvinoshandiswa kusunga block uye kugadzira chisimbiso chisina mhepo.
Bhodhi redunhu rakadhindwa (PCB) rakagadzirirwa kubatanidzwa nehurongwa hweC-PACE-EM. Masoketi ekubatanidza machine eSwiss ari paPCB akabatanidzwa kumaelectrodes egraphite newaya dzemhangura dzakaputirwa nesirivha uye screws dzebronze 0-60 dzakasungirirwa mumaelectrodes. Bhodhi redunhu rakadhindwa rinoiswa muvharo reprinta ye3D.
Mudziyo weCTCM unodzorwa ne programmable pneumatic actuator (PPD) inogadzira kumanikidzwa kweropa kwakarongwa kwakafanana nekutenderera kwemoyo. Sezvo kumanikidzwa mukati mekamuri yemhepo kuchiwedzera, silicone membrane inochinjika inokura kumusoro, ichimanikidza medium pasi penzvimbo yenyama. Nzvimbo yenyama inozotambanudzwa nekubuda kwemvura uku, zvichitevedzera kukura kwemuviri kwemoyo panguva ye diastole. Pakupera kwekureruka, kukurudzira kwemagetsi kwakaitwa kuburikidza nema graphite electrodes, izvo zvakaderedza kumanikidzwa mukamuri yemhepo uye zvakakonzera kusimba kwezvikamu zvenyama. Mukati mepombi mune vharuvhu yehemostatic ine sensor yekumanikidza yekuona kumanikidzwa muhurongwa hwemhepo. Kumanikidzwa kunonzwa ne sensor yekumanikidza kunoiswa kune data collector yakabatana nelaptop. Izvi zvinobvumira kutarisa kumanikidzwa kuri mukati mekamuri yegesi nguva dzose. Kana kumanikidzwa kwakanyanya kwekamuri kwasvika (standard 80 mmHg, 140 mmHg OS), mudziyo wekutora data wakarairwa kutumira chiratidzo kune C-PACE-EM system kuti igadzire chiratidzo che voltage ye biphasic ye 2 ms, yakaiswa ku 4 V.
Zvikamu zvemoyo zvakatorwa uye mamiriro ekurima mumigodhi mitanhatu akaitwa seizvi: Kutamisa mwoyo yakakohwewa kubva mumudziyo wekutamisa kuenda mutireyi ine cardioplegia inotonhora (4° C.). Ventricle yekuruboshwe yakabviswa nebanga risina kuchena ndokuchekwa kuita zvidimbu zve 1-2 cm3. Mabhuroko enyama aya akabatanidzwa pakutsigira kwenyama ne tissue adhesive ndokuiswa mubhavhu re microtome tissue rine mhinduro yeTyrode uye rinogara richiiswa oxygen (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), 10 mM HEPES (2.38 g), 1 mM MgCl2 (1 ml 1 M solution), 1.8 mM CaCl2 (1.8 ml 1 M solution), kusvika ku 1 L ddH2O). Microtome inodedera yakagadzirirwa kucheka zvidimbu zvine ukobvu hwe300 µm pa frequency ye80 Hz, amplitude yekudedera yakatwasuka ye2 mm, uye advanced rate ye0.03 mm/s. Tishu bath yakakomberedzwa nechando kuti mushonga ugare uchitonhorera uye tembiricha yakaramba iri pa4°C. Tumira zvikamu zvetishu kubva mu microtome bath kuenda ku incubation bath ine mhinduro yeTyrode inoenderera mberi neoxygen pachando kusvika zvikamu zvakakwana zve culture plate imwe chete zvawanikwa. Kune transwell cultures, zvikamu zvetishu zvakabatanidzwa kune polyurethane supports dzakachena dze 6 mm dzakafara uye dzakaiswa mu 6 ml ye optimized medium (199 medium, 1x ITS supplement, 10% FBS, 5 ng/ml VEGF, 10 ng/ml FGF-alkaline uye 2X antibiotic-antifungal ). Kukurudzira kwemagetsi (10 V, frequency 1.2 Hz) kwakaiswa kuzvikamu zvetishu kuburikidza neC-Pace. Panyaya dzeTD, T3 itsva neDex zvakawedzerwa pa100 nM uye 1 μM pakuchinja kwepakati pese. Nzvimbo yacho inozadzwa neokisijeni isati yatsiviwa katatu pazuva. Zvikamu zvenyama zvakakurirwa mu incubator pa37°C uye 5% CO2.
Kune CTCM cultures, zvikamu zvenyama zvakaiswa paprinta yakagadzirwa ne3D mudhishi rePetri rine mhinduro yakagadziridzwa yeTyrode. Mudziyo uyu wakagadzirirwa kuwedzera saizi yechidimbu chemoyo ne25% yenzvimbo yerin'i inotsigira. Izvi zvinoitwa kuitira kuti zvikamu zvemoyo zvisatambanuke mushure mekutamiswa kubva kumhinduro yeTyrode kuenda kune yepakati uye panguva yediastole. Uchishandisa guruu rehistoacrylic, zvikamu zvine ukobvu hwe300 µm zvakasungirirwa parin'i inotsigira ine dhayamita ye7 mm. Mushure mekubatanidza zvikamu zvenyama kune rin'i inotsigira, cheka zvikamu zvenyama zvakawandisa woisa zvikamu zvenyama zvakabatanidzwa mubhavhu reTyrode solution pachando (4°C) kusvika zvikamu zvakakwana zvagadzirwa zvemudziyo mumwe chete. Nguva yese yekugadzirisa yemidziyo yese haifanirwe kudarika maawa maviri. Mushure mekunge zvikamu zvitanhatu zvenyama zvabatanidzwa kune rings dzavo dzekutsigira, mudziyo weCTCM waunganidzwa. Kamuri rekudzidzisa reCTCM rinozadzwa ne21 ml pre-oxygenated medium. Tumira zvikamu zvenyama kuimba yekurima uye bvisa zvinyoro nyoro chero mabubble emhepo uchishandisa pipette. Chikamu chetishu chinozotungamirirwa kupinda mugomba uye chinodzvanywa zvinyoro nyoro panzvimbo yacho. Pakupedzisira, isa chivharo che electrode pachishandiso wobva waendesa mudziyo wacho ku incubator. Wobva wabatanidza CTCM nechubhu yemhepo uye sisitimu yeC-PACE-EM. Pneumatic actuator inovhurika uye vharuvhu yemhepo inovhura CTCM. Sisitimu yeC-PACE-EM yakagadzirirwa kupa 4 V pa1.2 Hz panguva yekufamba kwe biphasic kwe 2 ms. Medium yakachinjwa kaviri pazuva uye ma electrode akachinjwa kamwe chete pazuva kudzivirira kuungana kwe graphite pama electrodes. Kana zvichidikanwa, zvikamu zvetishu zvinogona kubviswa kubva mumatsime avo ekukudziridza kuti vaburitse chero mabubble emhepo angave akawira pasi pawo. Pamamiriro ekurapa kweMT, T3/Dex yakawedzerwa itsva nekuchinja kwega kwega kwepakati ne 100 nM T3 uye 1 μM Dex. Midziyo yeCTCM yakakurirwa mu incubator pa 37°C uye 5% CO2.
Kuti pave nematanho akatambanudzwa ezvidimbu zvemoyo, sisitimu yekamera yakakosha yakagadzirwa. Kamera yeSLR (Canon Rebel T7i, Canon, Tokyo, Japan) yakashandiswa neNavitar Zoom 7000 18-108mm macro lens (Navitar, San Francisco, CA). Kuona kwakaitwa patembiricha yemukamuri mushure mekutsiva chinhu chacho nechinhu chitsva. Kamera yakaiswa pakona ye51° uye vhidhiyo inorekodhwa pamafuremu makumi matatu pasekondi. Kutanga, software ye open source (MUSCLEMOTION43) yakashandiswa neImage-J kuyedza kufamba kwezvidimbu zvemoyo. Mask yakagadzirwa uchishandisa MATLAB (MathWorks, Natick, MA, USA) kutsanangura nzvimbo dzinofarirwa dzekurova zvidimbu zvemoyo kudzivirira ruzha. Mask dzakakamurwa nemaoko dzinoiswa pamifananidzo yese mufuremu uye dzobva dzaendeswa kuMUSCLEMOTION plug-in. Muscle Motion inoshandisa avhareji yesimba remapikiseli mufuremu yega yega kuyedza kufamba kwayo zvichienderana nefuremu rekutarisa. Data rakatorwa, rakaseferwa uye rakashandiswa kuyera nguva yekutenderera uye kuongorora kutambanudza kwenyama panguva yekutenderera kwemoyo. Vhidhiyo yakarekodhwa yakagadziriswa mushure mekushandisa firita yedhijitari ye zero-phase yekutanga. Kuti kuyera kutambanudza kwenyama (peak-to-peak), ongororo ye peak-to-peak yakaitwa kuti isiyanise pakati pe peaks ne troughs muchiratidzo chakarekodhwa. Pamusoro pezvo, detrending inoitwa uchishandisa 6th order polynomial kubvisa signal drift. Program code yakagadzirwa muMATLAB kuti ione kufamba kwenyama pasi rose, nguva yekutenderera, nguva yekuzorora, uye nguva yekudzikira (Supplementary Program Code 44).
Pakuongorora kusvuta, tichishandisa mavhidhiyo akafanana akagadzirwa ekuongorora kutambanudza kwemuchina, takatanga tatsvaga mifananidzo miviri inomiririra mafambiro epamusoro (nzvimbo dzepamusoro (dzepamusoro) nedzepasi (dzepasi) dzekufamba) zvichienderana nesoftware yeMUSCLEMOTION. Takazoparadzanisa nzvimbo dzematishu ndokushandisa fomu ye shading algorithm kune tissue yakakamurwa (Supplementary Fig. 2a). Tishu yakakamurwa kuita nzvimbo gumi dziri pasi, uye kushushikana pamusoro pega pega kwakaverengerwa uchishandisa equation inotevera: Strain = (Sup-Sdown)/Sdown, uko Sup neSdown ari madaro echimiro kubva pamusoro nepazasi pejira, zvichiteerana (Supplementary Fig. .2b).
Zvikamu zvemoyo zvakagadziriswa mu4% paraformaldehyde kwemaawa makumi mana nemasere. Nyama dzakagadziriswa dzakabviswa mvura mu10% ne20% sucrose kweawa imwe chete, dzozoiswa mu30% sucrose husiku hwese. Zvikamu zvakazoiswa muoptimum cutting temperature compound (OCT compound) uye zvishoma nezvishoma zvakaomeswa nechando mu isopentane/dry ice bath. Chengeta ma OCT embedding blocks pa -80 °C kusvika zvaparadzaniswa. Masiraidhi akagadzirwa sezvikamu zvine ukobvu hwe8 μm.
Kuti ubvise OCT kubva muzvikamu zvemoyo, pisa masiraidhi pamudziyo wekupisa pa95 °C kwemaminitsi mashanu. Wedzera 1 ml PBS kune imwe neimwe slide woiisa kwemaminitsi makumi matatu patembiricha yemukamuri, wozopinda muzvikamu nekuisa 0.1% Triton-X muPBS kwemaminitsi gumi nemashanu patembiricha yemukamuri. Kuti udzivise ma antibodies asiri chaiwo kuti asabatana nemuenzaniso, wedzera 1 ml ye3% BSA solution kumasiraidhi woiisa kweawa imwe patembiricha yemukamuri. BSA yakabva yabviswa uye masiraidhi akagezwa nePBS. Maka muenzaniso wega wega nepenzura. Ma antibodies ekutanga (akaderedzwa 1:200 mu 1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) uye troponin-T (Thermo Scientific; #MA5-12960) akawedzerwa kwemaminitsi makumi mapfumbamwe, uye ma antibodies echipiri (akaderedzwa 1:200 mu 1% BSA) achirwisa gonzo Alexa Fluor 488 (Thermo Scientific; #A16079), achirwisa tsuro Alexa Fluor 594 (Thermo Scientific; #T6391) kwemamwe maminitsi makumi mapfumbamwe. Takagezwa katatu nePBS. Kuti tisiyanise mavara akananga kubva kumashure, takashandisa antibody yechipiri chete senzira yekudzora. Pakupedzisira, mavara enyukireya eDAPI akawedzerwa uye masiraidhi akaiswa muvectashield (Vector Laboratories) ndokuvharwa ne nail polish. -x magnification) uye Keyence microscope ine 40x magnification.
WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) pa5 μg/ml muPBS yakashandiswa pakupenda WGA uye yakaiswa pazvikamu zvakagadzika kwemaminitsi makumi matatu patembiricha yemumba. Masiraidhi akazogezwa nePBS uye Sudan black yakawedzerwa pane slide yega yega uye yakachengetwa kwemaminitsi makumi matatu. Masiraidhi akazogezwa nePBS uye vectashield embedding medium yakawedzerwa. Masiraidhi akaoneswa paKeyence microscope pa40x magnification.
OCT yakabviswa kubva mumienzaniso sezvakatsanangurwa pamusoro apa. Mushure mekubvisa OCT, nyika masiraidhi mumushonga weBouin husiku hwese. Masiraidhi acho akazosukurudzwa nemvura yakasanganiswa kweawa imwe chete uye ozoiswa mumushonga weBibrich aloe acid fuchsin kwemaminitsi gumi. Zvadaro masiraidhi acho akasukwa nemvura yakasanganiswa ndokuiswa mumushonga we5% phosphomolybdenum/5% phosphotungstic acid kwemaminitsi gumi. Pasina kusukurudzwa, isa masiraidhi acho zvakananga mumushonga weaniline blue kwemaminitsi gumi nemashanu. Zvadaro masiraidhi acho akasukurudzwa nemvura yakasanganiswa ndokuiswa mumushonga we1% acetic acid kwemaminitsi maviri. Masiraidhi acho akaomeswa mu200 N ethanol ndokuiswa muxylene. Masiraidhi ane mavara akaoneswa uchishandisa maikorosikopu yeKeyence ine chinangwa che10x. Fibrosis area percentage yakayerwa uchishandisa Keyence Analyzer software.
CyQUANT™ MTT Cell Viability Assay (Invitrogen, Carlsbad, CA), nhamba yekatarogu V13154, zvichienderana neprotocol yemugadziri pamwe nekugadziriswa kwakati. Kunyanya, kuvhiyiwa kwekurova nedhayamita ye6 mm kwakashandiswa kuona kuti tissue yakakura zvakaenzana panguva yekuongorora MTT. Matumba acho akaiswa mumigodhi yeplate ine matsime gumi nemaviri ine MTT substrate zvichienderana neprotocol yemugadziri. Zvikamu zvacho zvinoiswa muicubation pa37° C. kwemaawa matatu uye tissue mhenyu inosanganisa MTT substrate kuti igadzire compound yepepuru formazan. Tsiva mhinduro yeMTT ne1 ml DMSO woiisa muicubation pa37°C kwemaminitsi gumi nemashanu kuti ubvise purple formazan kubva muzvikamu zvemoyo. Mienzaniso yakasanganiswa ne1:10 muDMSO mumaplate epasi ane 96-well clear uye purple color intensity yakayerwa pa570 nm uchishandisa Cytation plate reader (BioTek). Kuverengwa kwacho kwakajairwa kusvika pahuremu hwechikamu chimwe nechimwe chemoyo.
Chidimbu chemwoyo chakatsiviwa nechinhu chine 1 μCi/ml [5-3H]-glucose (Moravek Biochemicals, Brea, CA, USA) chekuyedza kushandiswa kweglucose sezvakatsanangurwa kare. Mushure memaawa mana ekuisira, wedzera 100 µl yepakati kune chubhu ye microcentrifuge yakavhurika ine 100 µl ye 0.2 N HCl. Zvadaro chubhu yacho yakaiswa muchubhu ye scintillation ine 500 μl ye dH2O kuti iome [3H]2O kwemaawa 72 pa37°C. Wobva wabvisa chubhu ye microcentrifuge kubva muchubhu ye scintillation wowedzera 10 ml ye scintillation fluid. Kuverengwa kwe scintillation kwakaitwa uchishandisa Tri-Carb 2900TR liquid scintillation analyzer (Packard Bioscience Company, Meriden, CT, USA). Kushandiswa kweglucose kwakazoverengerwa tichitarisa [5-3H]-shuga chaiyo, kusakwana kwechiyero uye mamiriro emuviri, kuderedzwa kwe [5-3H]-kusvika kushuga isina kunyorwa, uye kushanda kwe scintillation kunorwisa kushanda zvakanaka. Data iri rinoongororwa zvichienderana nehuremu hwezvikamu zvemoyo.
Mushure mekusanganiswa kwetishu muTrizol, RNA yakabviswa kubva muzvikamu zvemoyo ichishandisa Qiagen miRNeasy Micro Kit #210874 zvichienderana neprotocol yemugadziri. Kugadzirira, kutevedzana uye kuongorora data muRNAsec raibhurari kwakaitwa seizvi:
1 μg yeRNA pamuenzaniso mumwe nemumwe yakashandiswa senzira yekutanga kugadzirira raibhurari yeRNA. Maraibhurari ekutevera akaumbwa achishandisa NEBHext UltraTM RNA Library Preparation Kit yeIllumina (NEB, USA) zvichitevera mazano emugadziri, uye makodhi e index akawedzerwa kune attribute sequences yemuenzaniso wega wega. Muchidimbu, mRNA yakacheneswa kubva muRNA yese uchishandisa magineti beads akabatanidzwa ne poly-T oligonucleotides. Kupatsanurwa kunoitwa uchishandisa divalent cations pakupisa kwakanyanya muNEBHext First Strand Synthesis Reaction Buffer (5X). CDNA yekutanga yakagadzirwa uchishandisa random hexamer primers uye M-MuLV reverse transcriptase (RNase H-). CDNA yechipiri yakagadzirwa uchishandisa DNA polymerase I naRNase H. Ma overhangs asara anoshandurwa kuita blunt ends ne exonuclease/polymerase activity. Mushure mekuwedzerwa kwe3′ end yeDNA fragment, NEBHext Adapter ine hairpin loop structure inosungirirwa pairi kuti igadzirire hybridization. Pakusarudza zvidimbu zve cDNA zvine hurefu hwaidiwa, zvidimbu zve library 150-200 bp zvakacheneswa uchishandisa AMPure XP system (Beckman Coulter, Beverly, USA). Zvadaro, 3 μl USER Enzyme (NEB, USA) ine saizi yakasarudzwa cDNA yakasungirirwa ne adapter yakashandiswa kwemaminitsi gumi nemashanu pa37°C uye kwemaminitsi mashanu pa95°C PCR isati yasvika. PCR yakaitwa uchishandisa Phusion High-Fidelity DNA polymerase, universal PCR primers, uye Index (X) primers. Pakupedzisira, zvigadzirwa zve PCR zvakacheneswa (AMPure XP system) uye mhando ye library yakaongororwa paAgilent Bioanalyzer 2100 system. Library ye cDNA yakazorongwa uchishandisa Novaseq sequencer. Mafaira emifananidzo asina kuchena kubva kuIllumina akachinjwa kuita ma raw reads achishandisa CASAVA Base Calling. Data risina kuchena rinochengetwa mumafaira eFASTQ(fq) ane ma read sequences uye hunhu hwe base hunoenderana. Sarudza HISAT2 kuti ienderane ne filtered sequencing reads ne Sscrofa11.1 reference genome. Kazhinji, HISAT2 inotsigira genomes yehukuru hwese, kusanganisira genomes yakakura kupfuura mabhiriyoni mana emabhesi, uye default values ​​​​dzakagadzirirwa akawanda ma parameter. Splicing reads kubva kuRNA Seq data inogona kurongeka zvinobudirira uchishandisa HISAT2, system inokurumidza iripo parizvino, nekururama kwakafanana kana kuri nani kupfuura chero imwe nzira.
Kuwanda kwezvinyorwa zvinoratidza zvakananga mwero wekutaura kwemajini. Mazinga ekutaura kwemajini anoongororwa nehuwandu hwezvinyorwa (sequencing count) zvine chekuita negenome kana exons. Huwandu hwekuverenga hunoenderana nemazinga ekutaura kwemajini, kureba kwemajini, uye kudzika kwesequencing. FPKM (zvidimbu pazviuru zve base pairs zve transcript sequenced pa miriyoni ye base pairs) zvakaverengerwa uye P-values ​​​​dze differential expression dzakaongororwa uchishandisa DESeq2 package. Takazoverenga false discovery rate (FDR) yeP value yega yega tichishandisa Benjamini-Hochberg method9 zvichibva pane yakavakwa mukati R-function "p.adjust".
RNA yakabviswa kubva muzvikamu zvemoyo yakashandurwa kuita cDNA nehuwandu hwe200 ng/μl uchishandisa musanganiswa weSuperScript IV Vilo Master kubva kuThermo (Thermo, cat. nhamba 11756050). Quantitative RT-PCR yakaitwa uchishandisa Applied Biosystems Endura Plate Microamp 384-well transparent reaction plate (Thermo, cat. nhamba 4483319) uye microamp optical adhesive (Thermo, cat. nhamba 4311971). Musanganiswa we reaction waive ne5 µl Taqman Fast Advanced Master mix (Thermo, cat # 4444557), 0.5 µl Taqman Primer uye 3.5 µl H2O yakasanganiswa patsime. Maitiro eQPCR akajairwa uye CT values ​​​​dzakayerwa uchishandisa Applied Biosystems Quantstudio 5 real-time PCR instrument (384-well module; product # A28135). Ma primer eTaqman akatengwa kubva kuThermo (GAPDH (Ss03375629_u1), PARP12 (Ss06908795_m1), PKDCC (Ss06903874_m1), CYGB (Ss06900188_m1), RGL1 (Ss06868890_m1), ACTN1 (Ss01009508_mH), GATA4 (Ss03383805_u1), GJA1 (Ss03374839_u1), COL1A2 (Ss03375009_u1 ), COL3A1 (Ss04323794_m1), ACTA2 (Ss04245588_m1) Ma CT values ​​​​emasample ese akajairwa ku housekeeping gene GAPDH.
Kuburitswa kweNT-ProBNP munhau kwakaongororwa uchishandisa NT-ProBNP kit (nguruve) (Cat. No. MBS2086979, MyBioSource) zvichienderana nemutemo wemugadziri. Muchidimbu, 250 µl yemuenzaniso wega wega uye standard yakawedzerwa kaviri mutsime rega rega. Mushure mekuwedzera muenzaniso, wedzera 50 µl yeAssay Reagent A mutsime rega rega. Zunza plate zvinyoro nyoro wovhara nesealant. Zvadaro mapiritsi akaiswa pa37°C kweawa imwe chete. Wobva wadira mvura mumhinduro wosuka matsime ka4 ne350 µl ye1X wash solution, uchiisa mvura mumhinduro yekuwachisa kwemaminitsi 1-2 nguva imwe neimwe. Wobva wawedzera 100 µl yeAssay Reagent B patsime rega rega wovhara nesealant yeplate. Piritsi rakazunguzirwa zvinyoro nyoro ndokuiswa pa37°C kwemaminitsi makumi matatu. Fukidza mvura mumudziyo wosuka matsime kashanu uchishandisa 350 µl yemvura yekushambidza 1X. Wedzera 90 µl yemvura yekushambidza mumudziyo wega wega wovhara ndiro. Isa plate pa37°C kwemaminitsi 10-20. Wedzera 50 µl Stop Solution mumudziyo wega wega. Plate yakayerwa ipapo ipapo uchishandisa Cytation (BioTek) plate reader set pa450 nm.
Kuongororwa kwesimba kwakaitwa kuti pasarudzwe saizi dzeboka idzo dzichapa simba re >80% rekuona shanduko chaiyo ye10% muparameter ine chiyero chekukanganisa che5% Type I. Kuongororwa kwesimba kwakaitwa kuti pasarudzwe saizi dzeboka idzo dzichapa simba re >80% rekuona shanduko chaiyo ye10% muparameter ine chiyero chekukanganisa che5% Type I. Анализ мощности был выполнен для выбора размеров групп, которые обеспечат >80% выполнен для выбора размеров групп, которые обеспечат >80% выполнен для выбора размеров групп, которые обеспечат >80% выполнен для выбора размеров групп, которые обеспечат >80% мощности для обнаружения 10% частотой ошибок типа I. Kuongororwa kwesimba kwakaitwa kuti pasarudzwe hukuru hwemapoka aizopa simba re >80% rekuona shanduko ye10% yemaparameter akazara ne5% Type I error rate.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 обнаружности размера группы, который обеспечил бы > 80% мощности для обнаружения 10% 5% частоты ошибок типа I. Kuongororwa kwesimba kwakaitwa kuti pasarudzwe saizi yeboka raizopa >80% simba rekuona shanduko ye10% yemaparameter uye chiyero chekukanganisa che5% type I.Zvikamu zvenyama zvakasarudzwa zvisina tsarukano nguva isati yasvika yekuyedza. Ongororo dzese dzakanga dzisingatariswe nemamiriro ezvinhu uye sampuro dzakazonyorwa chete mushure mekunge data rese raongororwa. GraphPad Prism software (San Diego, CA) yakashandiswa kuita ongororo dzese dzezviverengero. Pahuwandu hwese, p-values ​​dzakaonekwa sedzakakosha pa<0.05. Pahuwandu hwese, p-values ​​​​dzakaonekwa sedzakakosha pamitengo <0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Pahuwandu hwese, p-values ​​​​dzakaonekwa sedzakakosha pamitengo <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. Pahuwandu hwese, p-values ​​​​dzakaonekwa sedzakakosha pamitengo <0.05.T-test yeMudzidzi ane tailed Two-tailed yakaitwa padata nekuenzanisa kaviri chete. ANOVA yenzira imwe chete kana kuti mbiri yakashandiswa kuona kukosha pakati pemapoka akawanda. Pakuita bvunzo dze post hoc, kugadzirisa kwaTukey kwakashandiswa pakuenzanisa kwakawanda. Data reRNAsec rine zvinhu zvakakosha zvekuverenga pakuverenga FDR uye p.adjust sezvakatsanangurwa muchikamu cheMethods.
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