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 siRNA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-siRNA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。
Objective To construct with the siRNA expression lentiviral vector,to infect to chondrocytes and to identify its interference effect.Methods First to obtain four different types of siRNA 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,the expression of uPA-mRNA gene in cartilage cells by and expression level of the uPA protein in cartilage cells were examined.The effect of proliferation of chondrocytes were detected by CCK-8.Results The four types of vectors were transfected into the primary culturing cartilage cells.The infection rate can be as high as 85％ in the experiment when MOI =100.P1,P2,P3 and P4 were all capable of inhibiting expressing of the uPA gene and protein in chondrocytes.P2 had the highest silencing rate.The significant promotive effect of uPA-siRNA on the reproduction of chondrocytes was shown by CCK-8 test after infection 96 hours.Conclusions The most efficient targeting uPA-siRNA sequence was found after screening.The siRNA lentiviral vector can be transfected into the cartilage cells,and can further inhibit the expressing of the uPA gene and steadily,and enhance proliferation of chondrocytes.
出处
《基础医学与临床》
CSCD
北大核心
2014年第5期602-609,共8页
Basic and Clinical Medicine
基金
国家自然科学基金(30960387
81260453)
兵团医药卫生重点攻关项目(2013BA020)
兵团国际交流合作项目(2011BC004)
