吉林大学学报(医学版) ›› 2019, Vol. 45 ›› Issue (01): 33-38.doi: 10.13481/j.1671-587x.20190107

• 基础研究 • 上一篇    

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

王秀艳, 樊晨星, 赵千, 王博, 袁松, 李昆   

  1. 锦州医科大学附属第一医院检验科, 辽宁 锦州 121001
  • 收稿日期:2018-05-30 发布日期:2019-01-28
  • 通讯作者: 李昆,副主任检验师,硕士研究生导师(Tel:0416-4197022,E-mail:lklszlym@163.com) E-mail:lklszlym@163.com
  • 作者简介:王秀艳(1991-),女,山东省聊城市人,在读医学硕士,主要从事干细胞药理学方面的研究。

Induction of tanshinone ⅡA on differentiation of human placenta-derived mesenchymal stem cells into cardiomyocytes and its mechanism

WANG Xiuyan, FAN Chenxing, ZHAO Qian, WANG Bo, YUAN Song, LI Kun   

  1. Department of Laboratory, First Affiliated Hospital, Jinzhou Medical University, Jinzhou 121001, China
  • Received:2018-05-30 Published:2019-01-28
  • Contact: 国家自然科学基金资助课题(81303255);辽宁省科技厅自然科学基金资助课题(201602291) E-mail:lklszlym@163.com
  • Supported by:
    This work was financially supported by the NSFCJoint Foundation of Yunnan Province (No. U1502223), the National Natural Science Foundation of China (Nos. 21837003, 21778059 and 81773611), the Science and Technology Program of Yunnan Province (No. 2018ZF001).

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(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)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。

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(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 of β-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-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