慢病毒载体介导miR-146a转染大鼠骨髓间充质干细胞的实验研究

Investigation on transduction of miR-146a in rat bone marrow mesenchymal stem cells mediated by lentiviral vector

目的探讨微小RNA-146a(miR-146a)体外转染大鼠骨髓间充质干细胞(BMSCs)应用于基因治疗的可行性。方法体外分离、培养并鉴定BMSCs,采用具有高效转染非分裂期细胞的慢病毒载系统将miR-146a导入BMSCs中,采用绿色荧光蛋白(EGFP)荧光技术、实时荧光定量PCR(qPCR)、Western Blot技术分别检测转染率、miR-146a及白介素-1(IL-1)受体相关激酶1(IRAK1)和肿瘤坏死因子受体相关因子6(TRAF6)蛋白水平表达情况。结果经流式细胞技术鉴定第三代BMSCs中表达CD90、CD106,不表达CD45。慢病毒感染MSCs的感染复数为50最佳感染率可达80%。EGFP荧光表达自72 h时开始逐渐增强。转染的BMSCs中miR-146a高表达。结论 (1)通过贴壁筛洗法能够获取纯度较高的BMSCs。(2)通过携带miR-146a的慢病毒载体成功转染BMSCs,并下调靶蛋白IL-1 IRAK1和TRAF6。

Objective To investigate the feasibility of therapy using microRNA-146 a(miR-146 a) transfected into rat bone marrow mesenchymal stem cells(BMSCs) in vitro for. Methods BMSCs were isolated, cultured and identified in vitro. The Lentiviral vector system was utilized to introduce miR-146 a into BMSCs. Expression of miR-146 a and its downstream target proteins were detected by real-time quantitative PCR(qPCR) and Western blot. EGFP marker was used to determine the expression of miR-146 a after transection. Result Flow cytometry analysis showed that BMSCs were positive for CD90, CD106, and negative for CD45. BMSCs were infected with lentivirus at a multiplicity of infection(MOI) of 50 with optimal expression efficiency of 80%. EGFP marker was observed at 72 hours after transfection and then gradually enhanced. Conclusion(1)BMSCs can be obtained by adhcrence scvecning method.(2) The lentiviral vector carrying miR-146 a was successfully transfected into BMSCs and stably expressed, which provided a theoretical foundation for therapy of lumbar back pain caused by lumbar disc herniation.