天津医药

• 细胞与分子生物学 •    下一篇

表达microRNA-203 tough decoy的慢病毒载体构建及应用

刘涛1,王玉亮1,宋红丽1,付楠楠2,吴本娟2,沈中阳1   

  1. 1. 天津市第一中心医院
    2. 天津医科大学
  • 收稿日期:2014-02-24 修回日期:2014-06-20 出版日期:2014-10-15 发布日期:2014-10-15
  • 通讯作者: 刘涛

Construction and Application of Lentiviral Vectors Expressing microRNA-203 Tough Decoy

  • Received:2014-02-24 Revised:2014-06-20 Published:2014-10-15 Online:2014-10-15
  • Contact: Tao LIU

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摘要：

【摘要】目的  建立表达microRNA（miRNA）tough decoy（TuD）的慢病毒载体构建方法，并检测其对细胞内 miRNA表达水平及细胞表型的影响。方法  在慢病毒表达质粒pSIH1-H1-copGFP中通过两步克隆，首先构建 miRNA TuD通用骨架质粒，进一步构建特异性针对大鼠miR-203的TuD表达载体。在293T细胞中包装成慢病毒后，感染大鼠骨髓间充质干细胞（BM-MSCs），同时用pSIH1-H1-copGFP空质粒包装慢病毒，感染BM-MSCs作为对照。定量RT-PCR检测细胞中miR-203的水平变化，并用CCK-8法和Annexin V-PI双标记法分别检测BMMSCs 的活性和凋亡。结果 成功构建了基于pSIH1-H1-copGFP质粒的miR-203 TuD表达载体，并对其序列进行了验证。用表达miR-203 TuD的重组慢病毒感染BM-MSCs后第3、6、9天检测细胞内源性miR-203表达水平降低，同时细胞活性增高，凋亡比例降低，与对照组相比差异有统计学意义。结论 两步克隆法构建miRNA TuD表达载体是一种简便高效的方法，其表达产物能够有效抑制细胞内源性miRNA水平，同时影响细胞表型。

关键词: 间质干细胞, 慢病毒属, 细胞凋亡, 微RNAs, 细胞活性, 遗传载体

Abstract:

[Abstract] Objective   To establish method of constructing lentiviral vectors to express microRNA（miRNA） ''tough decoy''（TuD）and to detect the effects of the TuD on cellular endogenous miRNA level and cellular phenotypes. Methods Two-step cloning strategy was utilized to first generate a universal miRNA TuD frame vector，followed by constructing the TuD expression vector specially targeting miR- 203. The package of the recombinant lentivirus was performed in 293T cells. Then the rat bone marrow mesenchymal stem cells（BM-MSCs）were infected by the miR-203 TuD expression lentivirus. The pSIH1-H1-copGFP vector was also packaged and the BM-MSCs infected by this lentivirus were served as control. Endogenous miR-203 level in BM-MSCs was measured by quantitative RT-PCR，and cellular viability and apoptosis were detected by CCK-8 test and Annexin V-PI staining respectively. Results  The miR-203 TuD expression vector was successfully constructed and inserted sequence was validated. At the 3rd，6th and 9th days after in fected by the miR-203 TuD expression lentivirus，rat BM-MSCs exhibited a repressed endogenous miR-203 level. The miR-203 TuD also promoted viability and inhibited apoptosis of BM-MSCs. All these differences between miR-203 TuD group and control group were statistically significant. Conclusion  The two-step cloning method for the construction of miRNA TuD expression vector is simple and efficient. The miRNA TuD can effectively suppress the level of the target miRNA and affect cellular phenotypes.

Key words: mesenchymal stem cells, lentivirus, cell cycles, microRNAs, cellular viability, genetic vectors