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目的探讨心肌细胞促进胚胎干细胞(embryonic stem cells,ESCs)分化为心肌样细胞的诱导作用。方法收集小鼠3.5d胚龄的囊胚,将其培养在小鼠胚胎成纤维细胞饲养层上,4~5d后取内细胞团接种在饲养层上分离培养出ESCs。取3~5代ESCs,先将ESCs悬浮培养形成2~3d的拟胚体(embryoid bodies,EBs),再与新生大鼠心肌细胞共培养诱导向心肌细胞分化,相差显微镜下观察分化细胞的形态学变化,免疫细胞荧光技术检测心肌细胞特异性肌钙蛋白T(TnT)、а-肌动蛋白(а-Actin)的表达。结果诱导d3起可见自发性、有节律跳动的拟胚体出现,12d时共培养组约有93%的拟胚体出现节律性收缩,显著高于对照组,均表达心肌细胞特异性蛋白cTnT、а-actin,心肌细胞直接接触诱导组其分化比率达56.5%,分化的细胞形态较单一。结论心肌细胞与胚胎干细胞直接接触能促进胚胎干细胞向心肌细胞分化。
Objective To investigate the induction of cardiomyocyte-induced embryonic stem cells (ESCs) into cardiomyocyte-like cells. Methods The blastocysts aged 3.5 days were collected and cultured on the feeder layer of mouse embryonic fibroblasts. After 4 ~ 5 days, the inner cell masses were inoculated on the feeder layer to isolate and culture ESCs. ESCs were isolated and cultured for 3 ~ 5 passages. ESCs were first cultured in the form of 2 ~ 3d embryoid bodies (EBs) and then co-cultured with neonatal rat cardiomyocytes to induce cardiomyocyte differentiation. The differentiated cells were observed under a phase contrast microscope The changes of cardiomyocyte-specific troponin T (TnT) and а-actin (а-Actin) were detected by immunofluorescence staining. Results From the induction of d3, the spontaneous and rhythmic beating embryoid bodies appeared. At about 12 days, about 93% of the embryoid bodies in the co-culture group showed rhythmic contractions, which were significantly higher than those in the control group. Both of them expressed cardiomyocyte-specific protein cTnT, а-actin, cardiomyocyte direct contact induction group differentiation rate of 56.5%, differentiated cells form a single. Conclusion The direct contact between cardiomyocytes and embryonic stem cells can promote the differentiation of embryonic stem cells into cardiomyocytes.