Apelin13对人胚胎干细胞定向心肌细胞分化效率的影响

摘要

目的 探讨Apelin13对人胚胎干细胞(hESCs)定向心肌细胞分化效率的影响.方法 将hESCs单层培养后经EDTA消化传代,应用明确的诱导分化培养基对观察组(加Apelin13)和对照组(不加Apelin13)定向诱导hESCs向心肌细胞分化.于诱导第2天(中胚层阶段)、第4天(心肌分化初始阶段)、第7天(心肌分化末阶段)作为时间节点,采用实时荧光定量PCR检测两组Brachury T、Mesp1、NKx2.5、APJ mRNA表达水平,采用Western blotting检测两组Brachury T、Mesp1、NKx2.5、APJ、Nanog、Sox2蛋白表达;同时于诱导第7天采用细胞免疫荧光技术检测两组心肌细胞标志物肌钙蛋白T2(TNNT2)、α-辅肌蛋白(α-actinin)表达.结果 诱导第7天观察组hESCs定向心肌分化程度高于对照组,免疫荧光染色倒置显微镜下可清楚看到成熟心肌细胞的肌结及心肌细胞TNNT2、α-actinin表达.在3个诱导时间节点,观察组Brachury T、Mesp1、NKx2.5、APJ mRNA及其蛋白表达均高于对照组(P均<0.05),观察组hESCs标志蛋白Nanog、Sox2低于对照组(P均<0.05).结论 Apelin13可提高hESCs向心肌细胞的分化效率.%Objective To explore the effects of Apelin13 on the differentiation of human embryonic stems cells (hESCs) into myocardial cells.Methods Feeder-free cultured hESCs were passaged into Matrigel-coated plates after being dissociated by EDTA.The cells were cultured in the differentiation medium with 100 nM Apelin13 (experimental group) or without Apelin13 (control group).On day 2, 4, and 7 of induction, real-time fluorescent quantitative PCR (qRT-PCR)was performed to detect the mRNA levels of the myocardial marker genes, including Brachyury T, Mesp1, Nkx2.5, and APJ.Meanwhile, the protein expression levels of Brachyury T, Mesp1, Nkx2.5, APJ, Nanog, and Sox2 were determined by Western blotting.Furthermore, the expression of TNNT2 and α-actinin at the transcription and protein levels in the differentiated myocardial cells after 7 days of induction was determined by fluorescent quantitative PCR and immunofluorescence, respectively.Results During the differentiation process, the introduction of Apelin13 promoted hESC differentiation, most significantly on day 7.Compared with the control group, the qRT-PCR showed that Apelin13 significantly increased the mRNA levels of Brachury T and Mesp1 after 2 days of induced differentiation, and Nkx2.5 and APJ after 7 days of differentiation.Western blotting showed the similar results with increased expression of Brachury T, Mesp1, Nkx2.5, and APJ after differentiation for 2, 4, and 7 days, and significant decrease in the expression of Nanog and Sox2.The differentiated myocardial cells after 7 days of culture also showed obviously positive expression of TNNT2 and α-actinin.Furthermore, the qRT-PCR demonstrated that Apelin13-treated cells showed much higher levels of TNNT2 and α-actinin after 7 days of induced differentiation (all P<0.05).Conclusion Apelin13 can improve the differentiation efficiency of hESCs into myocardial cells.