The Biological Effects Of Lentivirus Mediated MMP-3shRNA And Fas-siRNA Transfection In Human Degenerative Nucleus Pulposus Cells In Vitro

Objective: To study the biological effects of MMP-3shRNA gene and Fas-shRNA gene transfection in human degenerative nucleus pulposus cells in vitro.Methods:a.The culture of human degenerative nucleus pulposus cells:Cell viability was observed by trypan blue staining;the cell morphology was observed by toluidine blue staining;b.The sequences of MMP-3shRNA and Fas-siRNA were constructed according to the mRNA sequence of human MMP-3 gene and Fas gene,and the LV3 vector was linked with the 293 T vector after enzyme digestion;c.Lentivirus infected degeneration nucleus pulposus cells,according to the different treatment methods,the experiment was divided into 5 groups: A-blank control group,B-AAV negative control group,C-AAV-MMP-3shRNA group,D-AAV-FasshRNA group,E-AAV-FasshRNAMMP3 shRNA group;d.After 72 hours of transfection,the cell proliferation were measured by CCK8,levels of MMP-3,Fas,type II collagen and proteoglycan mRNA were detected by fluorescence quantitative PCR in human degenerated nucleus pulposus cells,after 96 hours,the relative expression of type II collagen and glycoprotein were detected by Western Blot.Results:a.The degenerated nucleus pulposus cells need to be adhered to the culture dish for 4-5 days.The initial cells were round or short spindle,polygonal,cytoplasmic stretched process,then raised gradually elongation,after 10-14 days 90% cells into a fusion state,such as whirlpool or flame like,abundant cytoplasm,cytoplasmic with refractive index,there were a large oval nucleus,clear outline,visible 1-3 nucleolus;.b.During the primary culture,the survival rate of degenerated nucleus pulposus cells was above 97%,and the survival rate was between 92-96% in the first generation,second generation could still maintain about 90%,then survival decreased gradually;c.After 72 hours with fluorescent microscope detection restructuring slow virus infection of human degenerative nucleus pulposus cells GFP,BFP expression situation,results showed that each group degenerative nucleus pulposus cells have reached a high transfection efficiency;d.Compared with the blank control group,the expression of Fas,MMP-3,type II collagen and proteoglycan increased in Fas interference group,MMP3 interference group and MMP3+Fas double gene co transfection group(mRNA),and the effect of double gene group was better than single gene group;Compared with the blank control group,the group of Fas interference,MMP3 interference and MMP3 + Fas double gene transfection group jointly proteoglycan and Ⅱ collagen increased,and the double effect of gene transfection group together is better than the single gene group,the above result differences are statistically significant(P<0.05).conclusion:1.Interfereing the expression of Fas and MMP-3 genes in nucleus pulposus cells through gene silencing technique could effectively inhibit the apoptosis of nucleus pulposus cells,and double gene co transfection could be synergistic。2.RNAi mediated by lentivirus can significantly inhibit the expression of MMP-3 and Fas in human degenerative nucleus pulposus cells,and increase the expression of extracellular matrix in degenerative nucleus pulposus,and dual genes have synergistic effects.