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