丹参酮ⅡA对人胎盘间充质干细胞向心肌细胞分化的诱导作用及其机制

doi:10.13481/j.1671-587x.20190107

摘要/Abstract

摘要： 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 （0.1 mg·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 （0.1 mg·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；Western-blotting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0.1 mg·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12 d以前呈快速增长趋势，培养12 d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（t_5-aza=2.937，P_5-aza<0.05；t_TanⅡA=4.769，P_TanⅡA<0.05）、ANF （t_5-aza=3.728，P_5-aza<0.05；t_TanⅡA=5.912，P_TanⅡA<0.05）、cTnI （t_5-aza=3.623，P_5-aza<0.05；t_TanⅡA=7.153，P_TanⅡA<0.05）和GSK-3β（t_5-aza=2.995，P_5-aza<0.05；t_TanⅡA=5.420，P_TanⅡA<0.05）蛋白表达水平明显升高，β-catenin （t_5-aza=2.985，P_5-aza<0.05；t_TanⅡA=6.951，P_TanⅡA<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。

关键词: 丹参酮ⅡA, 人胎盘间充质干细胞, 细胞分化, 心肌细胞

Abstract: Objective: To investigate the induction of tanshinone ⅡA (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 10.0 mg·L^-1), and the nontoxic dose of TanⅡA(0.1 mg·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 (0.1 mg·L^-1) group.After culture for 20 d,the expressions of α-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)and β-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 TanⅡ A was more than 0.1 mg·L^-1, the cell survival rates were decreased with the increase of concentration; the cells in control group showed a rapid growth trend before 12 d, 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 TanⅡ A induction group were significantly decreased(P<0.05).The immunohistochemistry staining results showed that the cells in control group didn't express α-SCA,and the cells in 5-aza induction group and TanⅡ A induction group expressed α-SCA, especially in TanⅡ A induction group.Compared with control group, the expression levels of GATA4(t_5-aza=2.937,P_5-aza<0.05;t_TanⅡA=4.769,P_TanⅡA<0.05), ANF(t_5-aza=3.728,P_5-aza<0.05;t_TanⅡA=5.912,P_TanⅡA<0.05), cTnI(t_5-aza=3.623,P_5-aza<0.05;t_TanⅡA=7.153,P_TanⅡA<0.05)and GSK-3β(t_5-aza=2.995,P_5-aza<0.05;t_TanⅡA=5.420,P_TanⅡA<0.05)proteins in the cells in 5-aza induction group and TanⅡA induction group were significantly increased, and the expression levels of β-catenin(t_5-aza=2.985,P_5-aza<0.05;t_TanⅡA=6.951,P_TanⅡA<0.05)protein were significantly decreased;compared with 5-aza induction group,the expression levels of GATA4,ANF,and GSK-3β proteins 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.

Key words: tanshinone ⅡA, human placenta-derived mesenchymal stem cells, cell differentiation, cardiomyocytes

中图分类号:

Q813.11

王秀艳, 樊晨星, 赵千, 王博, 袁松, 李昆. 丹参酮ⅡA对人胎盘间充质干细胞向心肌细胞分化的诱导作用及其机制[J]. 吉林大学学报(医学版), 2019, 45(01): 33-38.

WANG Xiuyan, FAN Chenxing, ZHAO Qian, WANG Bo, YUAN Song, LI Kun. Induction of tanshinone ⅡA on differentiation of human placenta-derived mesenchymal stem cells into cardiomyocytes and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2019, 45(01): 33-38.

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