5-氮杂胞苷诱导大鼠心脏成纤维细胞向心肌样细胞分化进程相关基因的表达差异  

Differences in genes expression associated with rat cardiac fibroblasts differentiation into cardiomyocyte-like cells induced by 5-azacytidine

作　　者：常玉巧[1] 贾阳阳[1] 韩贝 郭志坤[1] CHANG Yu-qiao;JIA Yang-yang;HAN Bei;GUO Zhi-kun(He'nan Key Laboratory of Medical Tissue Regeneration,Xinxiang Medical University,He'nan Xinxiang453003,China)

机构地区：[1]新乡医学院河南省医用组织再生重点实验室,河南新乡453003

出　　处：《解剖学报》2020年第6期861-867,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金(81570268);新乡医学院博士科研基金(XYBSKYZZ201602)。

摘　　要：目的探讨5-氮杂胞苷(5-aza)诱导心脏成纤维细胞(CFs)向心肌样细胞分化5 d、7 d、14 d、21 d和28 d相关基因的表达差异。方法新生1~3 d SD大鼠12只,胰蛋白酶/胶原酶联合消化法分离培养新生大鼠CFs,采用CFs分子标记盘状结构域受体2(DDR2)并行细胞免疫荧光染色。含15μmol/L 5-aza心肌细胞诱导液培养第3代CFs,Real-time PCR检测心肌化诱导不同时间点相关基因vimentin、DDR2、Nanog、sox2、c-kit、sca-1、Tbx5、CD73、CD34、c Myc、klf4、Gata4、Oct4、Mef2c和心肌肌钙蛋白T(c Tn T)的表达差异,细胞免疫荧光检测心肌化诱导后5 d、7 d、14 d、21 d和28 d肌钙蛋白T(c Tn T)的表达。透射电子显微镜观察心肌化诱导28 d CFs的超微结构变化。结果 5-aza诱导CFs后3 d细胞生长速率减慢,部分细胞由三角形、梭形向圆形和杆状转变。诱导后7 d、14 d、21 d和28 d,与诱导前相比,DDR2表达稳定,vimentin在14 d表达下降,Nanog、c-kit、sox2和Tbx5伴随着心肌化进程呈现表达下降趋势,而CD73、Mef2c、CD34、Gata4、Oct4和c Tn T表达逐步增加,sca-1在7 d表达上升又下降,c Myc和klf4在14 d表达上升又下降。诱导后5 d少量细胞表达c Tn T,14~21 d c Tn T阳性细胞数明显增高,28 d表达c Tn T较多且趋于稳定。透射电子显微镜显示,CFs心肌化诱导28 d,细胞与细胞间出现端-端连接,细胞质内出现大量的肌原纤维,排列较为规律,但肌节不明显,H带、Z线和M线显示不清。结论 CFs经5-aza诱导,可向心肌样细胞分化,但不能形成成熟的心肌细胞。Objective To explore the differences in genes expression associated with rat cardiac fibroblasts( CFs differentiation into cardiomyocyte-like cells induced by 5-azacytidine( 5-aza) at 5,7,14,21 and 28 days. Methods CFs were isolated from twelve 1-3-day neonatal SD rats by using combined type Ⅰ collagenase and trypsin digestion method.Cell purity was determined by CFs molecular marker discoidin domain receptor 2( DDR2) immunofluorescence assay.Myocardial induction solution containing 15 μmol/L 5-aza was incubated with passage 3 of CFs. The expression differences of related genes including vimentin,DDR2,Nanog,sox2,c-kit,sca-1,Tbx5,CD73,CD34,c Myc,klf4,Gata4,Oct4,Mef2 c and cardiac troponin T( c Tn T) were detected by Real-time PCR at different induction time. Cell immunofluorescence for c Tn T was observed at day 5,7,14,21 and 28 after myocardial induction. Morphological ultrastructure of induced CFs at day 28 was observed by transmission electron microscopy. Results The cell growth rate was slowed down at 3 days after5-aza induction,and the morphology of some cells were transformed from triangular,spindle shaped to round and rod shaped. Compared with the CFs before induction,induced after 7,14,21 and 28 days,DDR2 had stable expression,while vimentin expression decreased at day 14. But Nanog,c-kit,sox2 and Tbx5 declined accompanied by myocardial process,while CD73,Mef2 c,CD34,Gata4,Oct4 and c Tn T gradually increased,sca-1 rose at 7 days and then falled,c Myc and klf4 increased at 14 days and declined. At day 5,there were a few c Tn T positive cells and increased significantly at day 14-21. c Tn T positive cells were stable at day 28 and positive cells had no obviously change cultured from 35 days to40 days. The cytoplasm appeared in a large amount of collagen fiber bundle,arranged regularly and rich mitochondria under transmission electron microscope. It showed end to end connection between induced fibroblasts. There were a large number of myofibrils in the cytoplasm. However,sarcomere was not obvious,and the H

关 键 词：5-氮杂胞苷 心脏成纤维细胞 心肌样细胞 基因表达 实时定量聚合酶链反应 大鼠 

分 类 号：Q28[生物学—细胞生物学]