慢病毒介导的过表达miR181a基因修饰的骨髓间充质干细胞系的建立  

Construction of the recombinant lentivirus mediated miR181a-overexpressing bone marrow mesenchymal stem cells

作　　者：吕玮琦 胡瑞雪 张思凡 李呼伦[1] 刘玉梅[1] 穆莉莉[1] 孙博[1] 孔庆飞[1] 王广友[1] 刘希君[1] Lyu Weiqi;Hu Ruixue;Zhang Sifan;Li Hulun;Liu Yumei;Mu Lili;Sun Bo;Kong Qingfei;Wang Guangyou;Liu Xijun(Department of Neurobiology,Harbin Medical University,Harbin 150081,China)

机构地区：[1]哈尔滨医科大学神经生物教研室,150081

出　　处：《国际免疫学杂志》2021年第3期239-244,共6页International Journal of Immunology

基　　金：国家自然科学基金(81430035,81870955,81771305,81774024);黑龙江省自然科学基金(LH2020H024)。

摘　　要：目的构建携带过表达miR181a的慢病毒载体,转染骨髓间充质干细胞(bone mesenchymal stem cells,BMSC),使基因修饰后的BMSC持续高水平表达miR181a。方法将miR181a质粒与三种助转质粒PRSV,PRRE,VSVG共同转染进293T细胞中,构建含有增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)的过表达miR181a的慢病毒表达载体。72 h后收获过表达miR181a的病毒液,转染BMSC。用嘌呤霉素筛选,得到能够长期稳定过表达miR181a的BMSC,即miR181a-BMSC。结果成功构建出携带miR181a的慢病毒载体,转染率超过95%。嘌呤霉素筛选BMSC,得到能够长期稳定过表达miR181a的BMSC,与BMSC组相比,miR181a-BMSC组中,miR181a的表达量明显增多,差异具有统计学意义(3.80比25.92,t=19.401,P<0.05)。提取BMSC和miR181a-BMSC的外泌体,纯化后miR181a-BMSC的外泌体内miR181a表达量明显增高,差异具有统计学意义(1.41比0.87,t=6.003,P<0.05)。结论成功构建出慢病毒介导的过表达miR181a基因修饰的骨髓间充质干细胞。Objective The lentiviral vector carrying miR181a was constructed and transfected into bone mesenchymal stem cells BMSC to maintain high level of miR181a expression.Methods The 293T cells were co-transfected with the miR181a plasmid and three auxiliary packaging plasmids PRSV,PRRE and VSVG to construct the miR181a-overexpression recombinant lentiviral vector containing the enhance green fluorescent protein(eGFP).After 72 hours,the virus was harvested and transfected into BMSC.By screening with puromycin,miR181a-BMSC with stable and long-term overexpression of miR181a was obtained.Results The lentiviral vector carrying miR181a was successfully constructed,and the transfection rate was more than 95%.Stable cell line miR181a-overexpressing BMSCs were successfully screened by puromycin.Compared with BMSC group,the expression of miR181a was significantly increased in miR181a-BMSC group(3.80 vs 25.92,t=19.401,P<0.05).The exosomes of BMSC and miR181a-BMSC were extracted,and the expression of miR181a in exosomes of miR181a-BMSC was significantly increased after purification(1.41 vs 0.87,t=6.003,P<0.05).Conclusion Recombinant lentivirus mediated miR181a-overexpressing BMSCs were successfully constructed.

关 键 词：慢病毒载体 miR181a 骨髓间充质干细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]