人硫氧还蛋白基因重组慢病毒载体转染人脐带间充质干细胞的实验研究  

Study of hucMSCs Transfected with Recombinant Lentiviruses Containing Trx-1 Gene

作　　者：王明辉[1] 徐曼[1] 张斌[1] 陈虎[1] 

机构地区：[1]军事医学科学院附属医院造血干细胞移植科,北京100071

出　　处：《生物技术通讯》2016年第3期362-366,共5页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2013AA020103);医院创新科研基金(ZD-2012-01)

摘　　要：目的:构建携带人硫氧还蛋白(Trx-1)基因的人脐带间充质干细胞(hucMSCs),并对其生物学特性进行初步检测,为后续的细胞与基因治疗奠定实验基础。方法:以空载体转染的hucMSCs和未转染的hucMSCs作为对照,用重组慢病毒以最佳MOI值感染huc MSCs,应用Western印迹检测细胞中Trx-1蛋白的表达情况,流式细胞术检测细胞的细胞周期、免疫表型,CCK-8测定细胞的生长曲线。结果:通过EGFP表达的阳性细胞数筛选其最佳MOI值为10;Western印迹检测显示各组细胞均能不同程度地表达Trx-1蛋白,并且实验组Trx-1表达明显增加;免疫表型及细胞周期检测发现转染后实验组细胞基本特性无明显变化,而生长曲线则提示转染后实验组细胞生长受到抑制。结论:构建了携带人Trx-1基因的huc MSCs并能稳定高表达Trx-1,且其细胞学特性无明显改变,为进一步的细胞与基因治疗提供了一种新的手段。Objective：To construct human umbilical cord mesenchymal stem cells（hucMSCs） expressing human thioredoxin（Trx-1） gene,and to test their biological properties preliminary in order to lay an experiment foundation for treatment with the cell and gene therapy means.Methods：hucMSCs infected with recombinant lentiviruses at the best MOI was designated as the experiment group,and those transfected with empty vector and nontransfected hucMSCs as control groups.Western blot was used to detect Trx-1 protein expression in cells;flow cytometry was used for cell cycle,immune phenotype;and CCK-8 assay for the cell growth curve.Results：The best MOI was 10 after screening.Western blot results showed that Trx-1 was positively expressed in all hucMSCs,while it was highly expressed in hucMSCs infected with recombinant lentiviruses.The immune phenotype and cell cycle had no significant changes between all groups in the basic features,while cells growth was inhibited after transfection as the growth curve showed.Conclusion：hucMSCs were effectively infected with recombinant lentiviruses and could express Trx-1 highly and stably with no significant change,which provides a new approach for further cell and gene therapy.

关 键 词：硫氧还蛋白 脐带间充质干细胞 慢病毒 

分 类 号：Q78[生物学—分子生物学]