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目的探讨抗坏血酸作为诱导剂诱导人胚胎生殖细胞(hEGC)体外分化为心肌细胞。方法取5～10周人胚胎生殖腺嵴,进行组织块体外培养,用直接悬浮法使hEGC形成拟胚体(EBs),用不同浓度抗坏血酸的培养基对其进行诱导分化。然后取不同时间的细胞做免疫细胞化学染色,鉴定细胞的心肌特异转录因子GATA-4和心肌肌钙蛋白T(cTnT)表达情况。结果抗坏血酸诱导hEGC分化为心肌细胞的最佳浓度为0.1mg/ml,诱导第3周时分化率达(85.2±1.8)%,显著高于不添加任何诱导剂的对照组。诱导后细胞形态变成梭形,4周时细胞突起相互连接成条索状,且排列方向趋于一致;诱导后1周即开始出现GATA-4弱表达,第3周时表达最强;诱导后2周,细胞内开始表达cTnT,3周强阳性表达,4周表达明显增强。结论抗坏血酸能够促进hEGC向心肌细胞分化,可用以建立体外诱导hEGC分化为心肌细胞的方法 。
Objective To investigate the in vitro differentiation of human embryonic germ cells (hEGCs) into cardiomyocytes induced by ascorbic acid. Methods Human embryonic gonadal cristae were harvested from 5 to 10 weeks and cultured in vitro. The hESCs were induced to form EBs by direct suspension method, and their differentiation was induced by different concentrations of ascorbic acid. Then take different time cells do immunocytochemical staining to identify the cells of cardiac transcription factor GATA-4 and cardiac troponin T (cTnT) expression. Results Ascorbic acid induced hEGC to differentiate into cardiomyocytes at an optimal concentration of 0.1 mg / ml and the differentiation rate reached (85.2 ± 1.8)% at the third week of induction, which was significantly higher than that of the control group without any inducer. After 4 weeks, the cell protrusions became connected with each other into a cord-like shape, and the arrangement direction tended to be the same. The weak expression of GATA-4 began 1 week after induction and the expression was the strongest at the third week. After 2 weeks, cTnT began to be expressed in the cells, strongly positive for 3 weeks, and significantly increased for 4 weeks. Conclusion Ascorbic acid can promote the differentiation of hEGC into cardiomyocytes and can be used to establish a method of inducing hEGC to differentiate into cardiomyocytes in vitro.