基础医学与临床 ›› 2014, Vol. 34 ›› Issue (5): 602-609.

• 研究论文 • 上一篇    下一篇

慢病毒介导uPA-siRNA重组表达载体的构建及促进兔软骨细胞增殖

史晨辉1,王维山1,李长俊1,张振东1,郭风劲2,陈安民2   

  1. 1. 新疆石河子大学医学院第一附属医院骨科
    2. 华中科技大学 同济医学院 同济医院 骨科
  • 收稿日期:2013-08-09 修回日期:2013-12-20 出版日期:2014-05-05 发布日期:2014-04-28
  • 通讯作者: 王维山 E-mail:wwsmc2002@sina.com
  • 基金资助:
    国家自然基金;国家自然基金

Construction of the Targeted uPA-shRNA Lentiviral Vector and Its Promotion on proliferation of rabbit chondrocytes

  • Received:2013-08-09 Revised:2013-12-20 Online:2014-05-05 Published:2014-04-28

摘要: 目的 构建筛选出靶向特异uPA-siRNA慢病毒表达载体,感染软骨细胞后观察其对软骨细胞增殖及代谢的影响。方法 根据siRNA原理设计、构建4对靶向兔uPA shRNA序列(P1、P2、P3和P4),用RT-PCR筛选出高效靶向的P2序列。各序列经慢病毒包装后,通过Lipofectamine 2000转染入兔软骨细胞后,用RT-PCR和Western blot法分别检测uPA-siRNA对细胞内uPA mRNA和蛋白表达水平的抑制效果,用CCK-8法检测uPA-siRNA对软骨细胞增殖的影响。结果 成功构建4对uPA-shRNA序列并筛选出用于后续实验的高效靶向P2序列,各序列经慢病毒载体包装并成功转染到原代软骨细胞中,在感染复数(MOI)为100时感染率达到85%以上。P1、P2、P3和P4均可抑制软骨细胞中uPA基因及蛋白的表达,但P2沉默效果最好,基因抑制率达到70%,感染后软骨细胞的增殖受到促进。结论 成功构建高效靶向uPA-siRNA慢病毒载体,证实其可稳定转染软骨细胞并高效抑制uPA基因表达并促进软骨细胞增殖。

关键词: siRNA, RNA干扰, 慢病毒载体, 骨关节炎, 增殖

Abstract: Objective To construct the siRNA expression Lentiviral vector for uPA gene,infect to chondrocytes and identify its interference effect. Methods First, going through designing, constructing, restriction enzyme digestion, transformation, PCR identification, positive clone sequencing and lentivirus packaging, to obtain four different types of shRNA sequence (P1, P2, P3 and P4) from the targeted uPA gene of New Zealand rabbit based on siRNA theory. Secondly, to transfect the primary culturing cartilage cells of the New Zealand rabbit with P1, P2, P3 and P4, to observe the infection rate under fluorescence microscope, exam the expression level of the uPA-mRNA gene in cartilage cells by using the RT-PCR, and assay the expression level of the uPA protein in cartilage cells by conducting the Western-blot technology. The effect of proliferation of chondrocytes were detected by CCK-8. Results Constructed four different types of uPA-shRNA lentiviral vectors successfully, and these four types of vectors were all able to be transfected into the primary culturing cartilage cells. The infection rate can be as high as 85% in the experiment when MOI=100. It was showed that P1, P2, P3 and P4 were all capable of inhibiting expressing of the uPA gene and protein in chondrocytes. Moreover, among these four different sequences, the P2 had the highest silencing rate, which was 70%. It was further approved that this study has the statistical significance (P<0.05) when analyzing the result together with the control group. The significant Promotive effect of uPA-siRNA on the reproduction of chondrocytes was observed as determined by CCK-8 after 96 hours infection. Conclusions The most efficient targeted uPA-shRNA sequence was found after screening,It is also strongly verified that the siRNA lentiviral vector can be transfected into the cartilage cells, and can further inhibit expressing of the uPA gene efficiently and steadily, and can enhance proliferation of chondrocytes.

Key words: siRNA, RNA interfering, Lentiviral Vector, osteoarthritis, proliferation