miR－30a影响MSCs的细胞周期及其对DCs的抑制作用

摘要

目的：间充质干细胞（ MSCs）是一群具有自我更新能力和多向分化潜能的多功能干细胞。已经有研究表明， miR－30a在一些免疫相关疾病的MSCs中的表达发生变化。但对于miR－30a如何调控MSCs影响其免疫调节功能还未有报道。本研究探究了miR－30a对MSCs的表型、活力、凋亡、周期和免疫调节功能的影响。方法：用混合酶法分离人脐带来源的间充质干细胞；用流式细胞仪分析了高表达miR－30a对MSCs表型的影响；用CCK－8法检测了miR－30a对MSCs的活力的影响；用Annexin V／PI双染法检测了高表达miR－30a后细胞凋亡的情况；用Q－PCR和蛋白质免疫印迹的方法检测了miR－30a对周期蛋白Cyclin E2（CCNE2）的影响；通过Targetscan预测软件分析miR－30a与CCNE2的靶向关系，并通过荧光素酶检测试验进行验证；另外通过MSCs与树突状细胞（ DCs）共培养探讨了高表达miR－30a的MSCs对DCs的成熟的影响。结果：高表达miR－30a不影响MSCs的表型、活力和凋亡，但能抑制CCNE2的表达，使细胞周期阻滞在G0／G1期；进一步通过检测DCs的CD40和CD86表达发现，与普通MSCs相比，高表达miR－30a的MSCs能明显抑制DCs的成熟。结论：miR－30a可能通过抑制CCNE2表达影响MSCs的细胞周期，且miR－30a能增强MSCs对DCs的免疫抑制作用。%Objective:Mesenchymal stem cells( MSCs) have self-renewal capacity and potential to differentiate into the cells.It was reported that the expression of miR-30a changed in some immune diseases.But it remains unclear the effect of miR-30a on the im-munoregulatory functions of MSCs.Here we studied the impact of miR-30a on the phenotype,cell viability,apoptosis,cell cycle and im-munoregulatory functions of MSCs.Methods: The mixed enzyme methods were used for the isolation of human umbilical cord MSCs.Flow cytometry(FCM)was used to investigate the effect of overexpressed miR-30a on the phenotype of MSCs.CCK-8 was used to examine the cell viability of miR-30a-overexpressed MSCs.Annexin V/PI was used for the detection of apoptosis of MSCs.Q-PCR and Western blot were used to investigate the effect of miR-30a on the expression of Cyclin E2( CCNE2).CCNE2 was one putative target of miR-30a predicted by Targetscan database.The effects of miR-30a-overexpressed MSCs on the maturation of dendritic cells(DCs)were determined.Results:Overexpression of miR-30a blocked the cell cycle of MSCs in the G0/G1 phase by inhibiting the expression of CCNE2,but did not affect the phenotype, cell viability and apoptosis of MSCs.When co-cultured with DCs, although MSCs down-regulated the expression of CD40 and CD86 on DCs,overexpression of miR-30a more significantly enhanced the suppressive impact of MSCs on the maturation of DCs.Conclusion: miR-30a affects the cell cycle of MSCs and enhances its immunosuppressive effect on DCs.