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 (0.1mg·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=2.937,P5-aza<0.05;t_(TanⅡA)=4.769,P_(TanⅡA)<0.05)、ANF (t5-aza=3.728,P5-aza<0.05;t_(TanⅡA)=5.912,P_(TanⅡA)<0.05)、cTnI (t5-aza=3.623,P5-aza<0.05;t_(TanⅡA)=7.153,P_(TanⅡA)<0.05)和GSK-3β(t5-aza=2.995,P5-aza<0.05;t_(TanⅡA)=5.420,P_(TanⅡA)<0.05)蛋白表达水平明显升高,β-catenin(t5-aza=2.985,P5-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�
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
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)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2019年第1期33-38,218,共7页
Journal of Jilin University：Medicine Edition
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)
关键词
丹参酮ⅡA
人胎盘间充质干细胞
细胞分化
心肌细胞
tanshinone IIA
human placenta-derived mesenchymal stem cells
cell differentiation
cardiomyocytes
