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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。
In order to screen and establish an effective method to differentiate cardiomyocytes from porcine amniotic fluid stem cells, porcine amniotic fluid stem cells were selected as research objects. 5-azacytidine and vitamin C (Vc) Embryoid bodies (EBs) formed by stem cells are induced to differentiate. Immunofluorescence, RT-PCR and transmission electron microscopy were used to detect the expression of myocardial specific markers in the beating cell mass. The results showed that the cardiomyocyte-inducing agent was added to the embryoid body formed by porcine amniotic fluid stem cells, and the rhythmic beating cell mass was observed after 10 days. T test showed that 0.1 mmol / L Vc plus 5 μmol / L 5-aza combined induction group The highest induction efficiency, up to 33%. Immunofluorescence results showed that beating cardiomyocytes expressed cytoskeletal α-actin and troponin Tnni3. RT-PCR detected beating cardiomyocytes, and found that cardiomyocyte-specific marker molecules TbX5, Gata4, α-MHC, Tnni3 were positive expression. With the help of transmission electron microscopy, the beating-like cell mass after the induction can clearly show the structure of myofilament, glycogen granules, glycogen pool and the like. 5-azacytidine and vitamin C can promote the induction of differentiation of porcine amniotic fluid stem cells into cardiomyocytes.