骨髓间充质干细胞体内诱导分化为心肌细胞

观察骨髓间充质干细胞(mesenchymal stem cells，MSCs)植入体内后，在心肌微环境诱导下分化为心肌细胞的能力。无菌条件下取出大鼠双侧股骨及胫骨，冲洗骨髓腔获得细胞，贴壁筛选法纯化MSCs，体外培养、扩增，4，6-二咪基-4-联苯基吲哚(4，6-diamidino-2-phenylindole，DAPI)标记细胞，注入结扎冠脉左前降支所致心肌梗塞模型鼠的心肌组织。在不同时间点处死大鼠，获取心肌组织，采用HE染色和电镜技术对植入MSCs进行形态学观察和超微结构检测，荧光免疫组化检测植入MSCs肌球蛋白重链(MHC)和心肌特异性抗原Cx43的表达，同时应用RT-PCR技术检测心脏早期发育基因NKx2．5、GATA-4的表达。结果发现细胞标记效率为100％，通过连续检测MSCs植入后细胞形态从无规则状态、幼稚细胞表型逐渐向成熟心肌细胞方向转化，植入细胞排列同正常肌纤维方向平行，且植入四周后电镜检测到闰盘的存在；两周出现MHC的表达，后随时间延长表达逐渐增强。四周出现Cx43的表达，以后表达稳定，RT-PCR检测NKx2．5、GATA-4在一天即出现弱表达，两周～三周时表达最强，以后强度逐渐减弱。结果表明MSCs在体内微环境条件下能够转化为心肌细胞。

Experimental Study on Cardiomyocytes in vivo Differentiated form Mesenchymal Stem Cells

To investigate the ability to differentiate in vivo into cardiomyocytes form mesenchymal stem cell (MSCs). MSCs were isolated from bilateral thighbones and tibias of Wistar rats, purified by adhesive-screening method expanded in culture in vitro and labeled with DAPI, then injected into the myocardium of the rat recipients with acute myocardial infarction model which were created by legation of left anterior ascending artery. At intervals myocardial specimens around injection site were obtained, sectioned and stained with hematoxylin and eosin and electron microscopy for studying morphological changes of implanted MSCs. myosin heavy chain (MHC) and cardiac-specific antigen Cx43 were detected by immunohistochemistry, cardiac early-developmental gene NKx2.5 and GATA-4 were detected through RT-PCR. We found almost all of MSCs were labeled with DAPI. Viable cells labeled with DAPI were identified in host myocardium at all time after implantation. Implanted MSCs showed the growth potential in myocardial environments. MSCs reorganized themselves from a disorder pattern around injection site to an order structure along the long axis of normal myofibers. Furthermore, labeled MSCs were in parallel with the native fibers and integrated fully with the native fibers. The intercalated discs were detected at 4th week after implantation. Implanted MSCs demonstrated myogenic differentiation with the expression of MHC at 2nd week and cardiomyocytes phenotypes with the expression of Cx43 at 4th week, meanwhile, cardiac early-developmental gene NKx2.5 and GATA-4 expression and its quantities changes with interval time point were detected using RT-PCR, They display NKx2.5 and GATA-4 expressed at 1day after MSCs implantation, their expression quantity were up to peak at 2-3 week and from then on diseased. The result indicated that in response to myocardial microenvironments implanted MSCs can differentiated into cardiomyocytes, which were confirmed in morphology, histology and cellular biology.