miR-378对异体移植过程中骨髓间充质干细胞存活的影响

Effects of miR-378 on survival of bone marrow mesenchymal stem cells during allotransplantation

目的:探讨miR-378对异体移植过程中骨髓间充质干细胞(BMSCs)存活的影响。方法:采用密度梯度离心法分离大鼠BMSCs(rBMSCs),流式细胞仪检测rBMSCs表面标志物CD105、CD45、CD34、CD29和CD44的表达情况,茜素红和碱性磷酸酶染色观察细胞成骨诱导分化能力。采用miR-378模拟物或抑制剂转染rBMSCs,Real-time PCR检测细胞中miR-378表达情况。使用大鼠异体血清(ARS)干预转染后的rBMSCs,采用甲基偶氮唑蓝(MTT)法检测细胞存活率,AnnexinⅤ/PI双染流式细胞术检测细胞凋亡,Western blot检测凋亡蛋白Caspase-3表达水平。Europium标记转染后rBMSCs进行大鼠尾静脉移植,观察活体内rBMSCs存活情况。结果:经鉴定所分离的细胞确定为rBMSCs。转染miR-378模拟物(或抑制剂)后,rBMSCs中miR-378的表达显著增加(或降低)(P<0.05)。ARS干预后,rBMSCs存活率显著降低(P<0.05),凋亡率显著升高(P<0.05),cleaved-caspase-3表达显著上调(P<0.05);miR-378过表达(或低表达)可显著提高(或降低)ARS干预下rBMSCs的存活率,降低(或增加)细胞凋亡率,下调(或上调)cleaved-caspase-3表达。尾静脉异体移植rBMSCs后,miR-378过表达(或低表达)可显著提高(或降低)rBMSCs在活体内的存活率(P<0.05)。结论:miR-378可显著提高异体移植过程中BMSCs的存活率,且抑制细胞凋亡。

Objective: To explore the effect of miR-378 on survival of bone marrow mesenchymal stem cells (BMSCs) during allotransplantation. Methods: The rat BMSCs (rBMSCs) were isolated by density gradient centrifugation,cell surface epitopes of CD105,CD45,CD34,CD29 and CD44 were analyzed by flow cytometry,and the ability of osteogenic differentiation were detected by alizarin red staining and alkaline phosphatase staining.rBMSCs were transfected with miR-378 mimic or inhibitor,the expression of miR-378 was detected intransfected cells by Real-time PCR.Allogeneic rat serum (ARS) was used to treat the transfected rBMSCs.And then,the cell viability was detected using MTT assay,the apoptosis of rBMSCs were evaluated by Annexin V/PI double staining,the expression of caspase-3 was determined using Western blot.Finally,the Europium labeled of rBMSCs was injected into rats to observe the survival of rBMSCs. Results: The isolated cells were identified as BMSCs.After rBMSCs were transfected with miR-378 mimics (or inhibitors),the expression of miR-378 in rBMSCs was significantly increased (or decreased)( P <0.05).After incubated with ARS,the survival rate of rBMSCs was significantly decreased( P <0.05),the apoptosis rate was significantly increased( P <0.05),and the expression of cleaved-caspase-3 was significantly up-regulated( P <0.05).Overexpression (or low expression) of miR-378 in rBMSCs significantly increased (or decreased) the survival rate,decreased (or increased) apoptotic rate,and down-regulated (or up-regulated) cleaved-caspase-3 expression under the conditions of co-incubation with ARS( P <0.05).Overexpression (or low express-ion) of miR-378 significantly increased (or decreased) the survival rate of rBMSCs in vivo by the tail vein( P <0.05). Conclusion: miR-378 can significantly increase the survival rate of BMSCs during allotransplantation,and inhibit it apoptosis.