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  

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

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作  者:王秀艳 樊晨星 赵千 王博 袁松 李昆 WANG Xiuyan;FAN Chenxing;ZHAO Qian;WANG Bo;YUAN Song;LI Kun(Department of Laboratory,First Affiliated Hospital,Jinzhou Medical University,Jinzhou 121001,China)

机构地区:[1]锦州医科大学附属第一医院检验科,辽宁锦州121001

出  处:《吉林大学学报:医学版》2019年第1期33-38,218共7页Journal of Jilin University: Med Ed

基  金: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).

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10.0mg·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20 d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0.1mg·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=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。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 10.0 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 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 0.1 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 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(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

关 键 词:丹参酮ⅡA 人胎盘间充质干细胞 细胞分化 心肌细胞 

分 类 号:Q813.11[生物学—生物工程]

 

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