猪羊水干细胞特异向跳动心肌细胞诱导分化

Differentiation of porcine amniotic fluid stem cells into the beating cardiomyocytes

为了筛选并建立一种由猪羊水干细胞向心肌细胞分化的有效方法,以猪羊水干细胞为研究对象,以5-氮胞苷(5-aza)和维生素C(Vc)为诱导剂,对猪羊水干细胞形成的类胚体(EBs)进行诱导分化。应用免疫荧光、RT-PCR、透射电镜技术检测跳动细胞团中心肌特异性标记的表达情况。结果显示,在猪羊水干细胞形成的类胚体中加入心肌细胞诱导剂,10 d后即见到节律性跳动的细胞团,t检验发现0.1 mmol/L Vc加5μmol/L 5-aza联合诱导组的诱导效率最高,达33%。免疫荧光结果显示跳动心肌细胞团表达细胞骨架蛋白α-actin和肌钙蛋白Tnni3。RT-PCR检测跳动心肌细胞团,发现心肌细胞特异性标记分子TbX5、Gata4、α-MHC、Tnni3均呈阳性表达。借助透射电镜观察诱导后的跳动样细胞团,能清晰可见其中的肌丝、糖原粒、糖原池等结构。说明5-氮胞苷和维生素C可以促进猪羊水干细胞向心肌细胞的诱导分化。

The aim of this research is to find an effective cardiomyocyte-induced method derived from porcine amniotic fluid stem cells（pAFS）.For cardiac differentiation,the cells were formed embryoid bodies（EBs） firstly,then cultured in induced-medium including 5-azacytidine（5-aza） and vitamin C（Vc）.We detected the specific markers of cardiomyocyte by immunocytochemistry,RT-PCR and transmission electron microscope.The results showed that some embryoid bodies beat rhythmically after 10 days of induction.Furthermore,analysis of t test revealed that the percentage of beating cardiomyocyte-like cell clusters was highest（33%） when induction using 0.1 mmol/L Vc and 5 μmol/L 5-aza.Immunocytochemistry analysis demonstrated that cardiomyocyte-like cell clusters expressed α-actin,Tnni3.RT-PCR analysis also illustrated that TbX5,Gata4,α-MHC and Tnni3 were expressed positive in cardiomyocyte-like cell clusters.Especially,we observed basic structures of myocardium,such as myofilament,glycogen granule and so on by transmission electron microscope.In conclusion,5-azacytidine and vitamin C could promote differentiation of pAFS into myocardium.