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

摘要:

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5 μmol/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。

Abstract:

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.