淫羊藿苷联合白藜芦醇促进诱导性多能干细胞向心肌细胞分化的作用及机制研究

Study on Effects and Mechanism of Icariin Combined with Resveratrol for Differentiation of Induced Pluripotent Stem Cells into Cardiomyocytes

目的研究淫羊藿苷和白藜芦醇联合应用是否可进一步提高诱导性多能干细胞(iPS)向心肌细胞分化的作用,探讨其可能的机制。方法CCK8法检测淫羊藿苷和白藜芦醇对iPS增殖的影响;分别加入不同浓度淫羊藿苷和白藜芦醇诱导iPS向心肌细胞分化,RT-qPCR检测TNNI3、Cx43、α-actinin心肌细胞标志物和Oct4干细胞标志物;在常规诱导基础上,加入0.05μmol/L淫羊藿苷和0.1μmol/L白藜芦醇联合诱导iPS,Western blot检测心肌肌钙蛋白T(cTnT)的表达,RT-qPCR检测miR-1和miR-133的表达。结果0.05μmol/L淫羊藿苷对iPS增殖无明显影响,0.1、1、10μmol/L淫羊藿苷均能促进iPS增殖,各浓度白藜芦醇均可抑制iPS增殖;不同浓度淫羊藿苷均可下调Oct4的表达,上调TNNI3、α-actinin和Cx43的表达;白藜芦醇可下调Oct4的表达,上调TNNI3、α-actinin和Cx43的表达。与常规诱导方法比较,添加淫羊藿苷和白藜芦醇联合处理可明显上调α-actinin mRNA、cTnT蛋白和miR-1的表达,显著下调miR-133的表达。结论淫羊藿苷和白藜芦醇联合诱导可进一步促进iPS向心肌细胞的分化,其机制可能与调控miR-1和miR-133的表达有关。

Objective To study whether the combination of icariin and resveratrol can further improve the effects of differentiation of induced pluripotent stem cells(iPS)into cardiomyocytes;To discuss its possible mechanism.Methods CCK8 assay was used to detect the effects of icariin and resveratrol on the proliferation of iPS.Different concentrations of icariin and resveratrol were chosen to induce the differentiation of iPS into cardiomyocytes,and RT-qPCR was used to detect the changes of cardiomyocyte markers TNNI3,Cx43,α-actinin and stem cell marker Oct4.On the basis of conventional induction,the combination of 0.05μmol/L icariin and 0.1μmol/L resveratrol were added to induce iPS;the expression of cTnT was detected by western blot;the expression of miR-1 and miR-133 were detected by RT-qPCR.Results 0.05μmol/L icariin had no significant effect on the proliferation of iPS.0.1μmol/L,1μmol/L and 10μmol/L icariin could promote the proliferation of iPS,while resveratrol could inhibit the proliferation of iPS.Different concentrations of icariin could down-regulate the expression of Oct4,but up-regulate the expressions of TNNI3,Cx43 andα-actinin;resveratrol also could decrease the expression of Oct4 and increase the expressions of TNNI3,Cx43 andα-actinin.Compared with the conventional induction method,the combination of icariin and resveratrol could significantly increase the expressions ofα-actinin mRNA,cTnT protein and miR-1,but significantly inhibit the expression of miR-133.Conclusion The combination of icariin and resveratrol can further promote the differentiation of iPS into cardiomyocytes,and the mechanism may be related to the regulation of expressions of miR-1 and miR-133.