UPA-siRNA Restructuring Lentiviral Vector Infected Rabbit Chondrocytes Preliminary Study On Its Multiplication

Objective: Construction of targeting specific urokinase-type plasminogen activator(u PA)-si RNA after lentiviral vector and screened efficient targeting sequences, transfected rabbit chondrocytes were observed for chondrocyte proliferation, u PA, MMP-3 gene and protein expression levels of influence.Methods:Take good growth in primary rabbit articular cartilage cells were passaged inoculation training, according to the gene sequences in Gen Bank rabbit u PA, si RNA target reference design principles,design, construct synthetic rabbit u PA-si RNA sequence targeting,using RT-PCR screening of efficient targeting sequence, according to different multiplicity of infection(MOI) value added Lentiviral Particles liquid,after co-culture to determine the optimal multiplicity of infection(MOI) value and virus infection efficiency measurement and analysis, efficient targeting sequence by lentiviral packaged by Lipofectamine 2000 transfected rabbit chondrocytes,according randomized experiment,divided into experimental group(u PA-si RNA transfected with lentiviral vector group),empty vector group(transfected with empty lentiviral vector group),control group(not any treatment group),drug control group(load MMP inhibitor TIMP). Cells in vitro culture 96 h,using real-time quantitative reverse transcription-polymerase chain reaction(RT-PCR)and Western blot(Western blot)were detected chondrocytes u PA-si RNA intracellular u PA,MMP-3 m RNA and protein expression levels,and by CCK-8 assay u PA-si RNA on chondrocyte proliferation.Results : Lentiviral vectors transfect primary chondrocytes by si RNA vector plasmid sequencing consistent with the expected si RNA sequence,DNA sequencing correct sequence without the mutation,screened out efficiently targeting sequence(ie silence best).After transfection,cells were cultured chondrocytes seen early cartilage cells were polygonal, adherent cells grow;when you pass two generations,showing spindle cells,and aggregation growth;when five passages,the cells showed a typical fibroblast-like;in line with the growth of cartilage cells Features,subsequent experiments can be carried out.With increasing multiplicity of infection(MOI),the efficiency of lentiviral infection also increases,when multiplicity of infection(MOI) of 100 when the infection rate of over 85%,in line with the follow-up experiments.Cartilage cell proliferation is detected by CCK-8 si RNA effect on chondrocyte proliferation was found to give u PA-si RNA transfection after lentiviral vector has not been suppressed.Experimental group u PA m RNA,MMP-3 m RNA and expression of u PA,MMP-3 protein levels were significantly lower than the empty vector rent,blank control group and drug group(P<0.05),while the empty vector group and blank control group and drug control Group u PA m RNA,MMP-3 m RNA and expression of u PA,MMP-3 protein level difference was not statistically significant(P>0.05).Conclusion:Successfully build efficient targeting uPA-siRNA lentiviral vectors, which can be stably transfected rabbit chondrocytes and can effectively inhibit the gene and protein expression u PA,but also on the MMP-3 gene and protein expression was inhibited,the chondrocyte proliferation play facilitating role.