【摘要】 目的:观察5-氮胞苷(5-aza)在体外诱导人骨髓间充质干细胞转化为心肌样细胞的心钠素(ANP)、脑钠素(BNP)、α-骨骼肌动蛋白(α-skeletal actin)、肌球蛋白轻链-2v(MLC-2v)、锌指结构转录因子(GATA-4)和心脏特异同源转录因子(Nkx2.5)基因表达。方法:取非血液疾病的5例胸科手术患者术中已切除掉的肋骨,抽取骨髓。利用淋巴细胞分离液行密度梯度离心法和差异贴壁法进行分离、提纯骨髓间充质干细胞,并进行培养扩增。用流式细胞仪对第2代的骨髓间充质干细胞细胞表面抗原进行测定。应用10μmol/L5-aza对第2代的骨髓间充质干细胞诱导24h,于诱导后1,2,3及4周,用反转录-聚合酶链反应检测ANP、BNP、α-skeletal actin、MLC-2v、GATA-4和Nkx2.5基因表达。结果:3份第2代骨髓间充质干细胞样本重复测定,表达CD29,CD44,不表达CD34,CD45。以5-aza诱导培养24 h后,骨髓间充质干细胞形态和排列方式发生明显变化,诱导1周后,细胞体积增大,多呈长梭行,平行排列;诱导2周后,细胞变为短柱状,突起位于2端,相邻细胞的突起紧密接触;诱导3周后,短柱状细胞的突起相互连接,部分细胞可见类肌管样结构;诱导4周后,细胞体积变小,可见由几个细胞连接形成的多核肌管样结构。诱导组细胞ANP、BNP、α-skeletal actin、MLC-2v、GATA-4和Nkx2.5的聚合酶链反应产物凝胶电泳呈阳性,对照组为阴性,诱导组基因表达量均随着时间延长逐渐增加。结论:5-aza可诱导骨髓间充质干细胞向心肌样细胞转化,其处于祖心肌细胞和分化的心肌细胞之间,并可能具有心肌细胞的功能。更多还原

【Abstract】 Objective:To study the data of differentiation of bone marrow derived from human mensenchymal cells(MSCs) induced with 5-azacytindine into cardiomyocytes and ANP,BNP,α-skeletal actin,MLC-2v,GATA-4 and Nkx2.5 expressing in cardiomyocytes.Methods:Bone marrow came from human ribs.The methods of density gradient centrifugation and adherent filtration were used to isolate and purify MSCs.The MSCs were cultured.MSCs surface antigen profiles obtained by flow cytometry.The MSCs of second passage were induced with 5-azacytindine at 10 μmol/L for 24 hours.ANP,BNP,α-skeletal actin,MLC-2v,GATA-4 and Nkx2.5 were tested by RT-PCR at first week,second week,third week and fourth week after the MSCs were treated with 5-azacytindine.Results:Three sample of MSCs of second passage were tested.The MSCs were positive for CD29,CD44 and negative for CD34,CD45.After the MSCs treated with 5-azacytindine 24 hours,the morphology of them changed obviously.After one week,the cells became larger,and most of them became long shuttle-shape and parallel.After two weeks,most of the cells became rod-shape,and there were umbos on the tow sides of the cells,and the umbos of near cells touched tightly.After three weeks,the near cells’ cytomembrane touched each other to form myotube-shape.After four weeks,the cells became small,and the multinucleus myotube-shape constructions were formed by a few cells.ANP,BNP,α-skeletal actin,MLC-2v,GATA-4 and Nkx2.5 were positive in MSCs treated with the drug and negative in control group.With the cells’culture time up,the levels of GATA4 and Nkx2.5 in MSCs were up.Conclusion:MSCs can induced into cardiomyocytes with 5-azacytindine.The stage of differentiation of the MSCs is between cardiomyocyte-progenitor and differentiated cardiomyocytes and the cells may be with fountion of cardiomyocyte.更多还原