齐墩果酸对hiPSCs衍生心肌细胞成熟的促进作用及机制

Effect and mechanism of oleanolic acid on myocardial maturation derived from hiPSCs

目的探讨齐墩果酸(OA)促进丙酮酸激酶(PK)同工型转换在人诱导多能干细胞(hiPSCs)衍生心肌细胞(hiPSC-CMs)成熟过程中的作用。方法通过时序性激活和抑制Wnt信号通路,诱导未分化hiPSCs向心肌细胞分化,在诱导第19天将hiPSC-CMs分为空白对照组(添加B27的RPMI 1640培养基)、DMSO组(添加DMSO 2μl/ml)、OA加药组(添加5 mmol/L的OA 2μl/ml),所有培养基每2 d更换1次,共处理7 d。免疫荧光及RT-qPCR检测hiPSCs中干性因子及hiPSC-CMs中心肌特异性标志物的表达情况;Western blotting检测各组PK、线粒体融合蛋白(MFN2)的表达情况;Mito-Tracker染色检测各组细胞线粒体形态,电镜观察各组线粒体超微结构;EDU染色检测各组细胞增殖情况;流式细胞仪检测各组细胞的细胞周期。结果免疫荧光染色结果显示,hiPSCs表达干性因子Nanog、Sox2,hiPSC-CMs表达心肌特异性标志物cTnT、Cx43、α-actinin。RT-qPCR检测结果显示,与诱导前的hiPSCs比较,hiPSC-CMs心肌相关基因TNNI3、MYH6、MYH7的表达明显上调,差异有统计学意义(P<0.05或P<0.01)。免疫荧光染色结果显示,与空白对照组、DMSO组比较,OA加药组细胞面积、肌节长度增加,细胞圆度指数降低,差异均有统计学意义(P<0.05)。Western blotting检测结果显示,与空白对照组、DMSO组比较,OA加药组丙酮酸激酶1与丙酮酸激酶2比值(PKM1/PKM2)及MFN2的表达明显增高,差异有统计学意义(P<0.05或P<0.01)。电镜观察见OA加药组线粒体融合变长;Mito-Tracker染色结果显示,OA加药组线粒体形成更成熟的网状结构。EDU染色结果显示,与空白对照组、DMSO组比较,OA加药组细胞增殖率降低,差异有统计学意义(P<0.05)。流式细胞仪检测结果显示,与空白对照组、DMSO组比较,OA加药组多核细胞数有所增加。结论OA可促进hiPSC-CMs细胞结构、线粒体结构及形态的成熟,也可促进细胞多核化,并可通过调节PK同工型转换促进hiPSC-CMs的成熟。

Objective To investigate the effect of oleanolic acid(OA)in the maturation of human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)through switching pyruvate kinase type M isoforms.Methods HiPSC-CMs were derived from human induced pluripotent stem cells(hiPSCs),through sequential activation and inhibition of the Wnt signaling pathway.On day 19 hiPSC-CMs were divided into control group(RPMI 1640+B27),DMSO group(RPMI 1640+B27+2μl DMSO),and 10μmol/L OA group(RPMI 1640+B27+2μl 5 mmol/L OA)and treated for 7 days,and the medium was changed every 2 days.Immunofluorescence or RT-qPCR were used to detect the expression of stem factor in hiPSCs and cardiac specific markers in hiPSC-CMs.Western blotting was used to detect the expression of PK and mitofusin 2(MFN2).Mito-Tracker staining showed the morphology of mitochondria,and transmission electron microscope(TEM)showed the ultrastructure of mitochondria.EDU staining was used to detect cell proliferation,and flow cytometry was used to detect cell cycle.Results Immunofluorescence showed that hiPSCs expressed Nanog and Sox2,and hiPSC-CMs expressed myocardial specific markers cTnT,Cx43 andα-actinin.RT-qPCR showed that,compared with hiPSCs,significant differences(P<0.05 or P<0.01)existed in hiPSC-CMs expressed troponin I3(TNNI3),myosin heavy chain 6(MYH6),and myosin heavy chain 7(MYH7).Immunofluorescence showed that,compared with control group and DMSO group,the cell area increased,sarcomere length increased,and the cell circularity decreased in hiPSC-CMs cultured with OA,and the differences were statistically significant(P<0.05);Western blotting showed that,compared with control group and DMSO group,the ratio of PKM1/PKM2 and the expression of MFN2 increased obviously,and the differences were statistically significant(P<0.05 or P<0.01),the fusion lengthening of mitochondria was observed under electron microscope,and Mito-Tracker staining showed that mitochondria formed more mature structure;EDU staining showed a decreased cell proliferation in hiPSC-CMs cultured with OA group compared with control group and DMSO group,all differences were statistically significant(P<0.05).The flow cytometry results indicated that the number of coenocytes increased in hiPSC-CMs cultured with OA group compared with control group and DMSO group.Conclusion OA can promote the maturation of cell structure,mitochondrial structure and morphology,and also can promote cell multinucleation.OA can promote hiPSC-CMs'maturation by switching pyruvate kinase type M isoforms.