Objective:To investigate the induction of tanshinoneⅡA (TanⅡA) on the differentiation of human placenta-derived mesenchymal stem cells (hPDMSCs) into cardiomyocytes, and to provide an experimental basis for TanⅡA as a cardiomyocyte differentiation inducer.Methods:The hPDMSCs were treated with different concentrations of TanⅡA (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0mg·L-1) , and the nontoxic dose of TanⅡA (0.1mg·L-1) was screened by MTT assay for experiment.The hPDMSCs were divided into control group, 5-aza induction (10μmol·L-1) group, and TanⅡA induction (0.1mg·L-1) group.After culture for 20d, the expressions ofα-sarcomeric actin (α-SCA) in the cells in various groups were detected with immunohistochemistry;the positive expression rates of cardiac troponin I (cTnI) in the cells in various groups were detected with immunofluorescence, and the differentation rates of cardiomyocytes were calculated.The expression levels of GATA-binding protein 4 (GATA4) , atrial natriuretic factor (ANF) , cTnI, glycogen synthase kinase-3β (GSK-3β) andβ-catenin in the cells were detected with Western blotting method.Results:The biological characteristics of hPDMSCs accorded with the mesenchymal stem cells.The MTT results showed that when the concentration of TanⅡA was more than 0.1mg·L-1, the cell survival rates were decreased with the increase of concentration;the cells in control group showed a rapid growth trend before 12d, and the proliferation activities of the cells began to decrease on the 12th day.Compared with control group, the cell activities in 5-aza induction group and TanⅡA induction group were significantly decreased (P＜0.05) .The immunohistochemistry staining results showed that the cells in control group didn't expressα-SCA, and the cells in 5-aza induction group and TanⅡA induction group expressedα-SCA, especially in TanⅡA induction group.Compared with control group, the expression levels of GATA4 (t5-aza=2.937, P5-aza＜0.05;tTanⅡA=4.769, PTanⅡA＜0.05) , ANF (t5-aza=3.728, P5-aza＜0.05;tTanⅡA=5.912, PTanⅡA＜0.05) , cTnI (t5-aza=3.623, P5-aza＜0.05;tTanⅡA=7.153, PTanⅡA＜0.05) and GSK-3β (t5-aza=2.995, P5-aza＜0.05;tTanⅡA=5.420, PTanⅡA＜0.05) proteins in the cells in 5-aza induction group and TanⅡA induction group were significantly increased, and the expression levels ofβ-catenin (t5-aza=2.985, P5-aza＜0.05;tTanⅡA=6.951, PTanⅡA＜0.05) protein were significantly decreased;compared with 5-aza induction group, the expression levels of GATA4, ANF, and GSK-3βproteins in TanⅡA induction group were increased (P＜0.05) .Conclusion:TanⅡA can induce the differentiation of hPDMSCs into cardiomyocytes, which has better effect than 5-aza, and its mechanism may be related to inhibiting the Wnt/β-catenin signaling pathway.%目的:探讨丹参酮ⅡA (TanⅡA) 对人胎盘间充质干细胞 (hPDMSCs) 向心肌细胞分化的诱导作用及其机制, 为TanⅡA作为心肌细胞分化诱导剂提供实验依据.方法:采用不同浓度TanⅡA (0.1、0.2、0.4、0.6、0.8、1.0、2.0、4.0、6.0、8.0和10.0 mg·L-1) 处理hPDMSCs, MTT法筛选出无毒剂量的TanⅡA (0.1mg·L-1) 用于实验.hPDMSCs培养体系分为对照组、5-氮胞苷 (5-aza, 10μmol·L-1) 诱导组和TanⅡA (0.1mg·L-1) 诱导组.培养20d后, 免疫组织化学法检测各组细胞中α-横纹肌肌动蛋白 (α-SCA) 的表达;免疫荧光法检测各组细胞中心肌肌钙蛋白I (cTnI) 的阳性表达率, 计算心肌细胞分化率;Westernblotting法检测各组细胞中心肌转录调节因子4 (GATA4) 、心钠素 (ANF) 、cTnI、糖原合成酶激酶-3β (GSK-3β) 和β-连环蛋白 (β-catenin) 的蛋白表达水平.结果:hPDMSCs的生物学特性符合间充质干细胞.MTT法, 当TanⅡA浓度大于0.1mg·L-1时, 细胞存活率随浓度的增加而降低.对照组细胞在培养12d以前呈快速增长趋势, 培养12d后细胞增殖活性降低;与对照组比较, 5-aza诱导组和TanⅡA诱导组细胞增殖活性明显降低 (P＜0.05) .免疫组织化学染色, 对照组细胞不表达α-SCA, 5-aza诱导组和TanⅡA诱导组细胞均表达α-SCA, 且TanⅡA诱导组更明显.与对照组比较, 5-aza诱导组和TanⅡA诱导组细胞中GATA4 (t5-aza=■0.05) 、cTnI (t5-aza=3.623, P5-aza＜0.05;tTanⅡA=7.153, PTanⅡA＜0.05) 和GSK-3β (t5-aza=2.995, P5-aza＜0.05;tTanⅡA=5.420, PTanⅡA＜0.05) 蛋白表达水平明显升高, β-catenin (t5-aza=2.985, P5-aza＜0.05;tTanⅡA=6.951, PTanⅡA＜0.05) 蛋白表达水平明显降低;与5-aza诱导组比较, TanⅡA诱导组GATA4、ANF和GSK-3β蛋白表达水平进一步升高 (P＜0.05) .结论:TanⅡA能诱导hPDMSCs分化为心肌细胞, 且效果优于5-aza, 其机制可能与TanⅡA能抑制Wnt/β-catenin信号通路有关.
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