Study Of Lentivirus-mediated Sox9 Overexpression And Fas Interferon Transfection Of Human Degenerative Nucleus Pulposus Cells In Vitro

Objective To recover the cryopreserved degenerative human nucleus pulposus cells and observe their viability.To screen the optimal Fas-si RNA in vitro and its lentiviral vector was constructed.To study the influence on human degenerative nucleus pulposus cells transfectd with Sox9,Fas and Sox9 combining with Fas in vitro.Methods Recover and culture the human degenerative disc nucleus pulposus cells storaged in liquid nitrogen and observe its vitality.The recombinant lentiviral vector was designed by Fas-si RNA and the recombinant clones were identified by restriction enzyme digestion and gene sequencing.Lentivirus packaging on Fas-si RNA and detecting its titer.Sox9 overexpression and Fas interference were used to transfect human degenerateive nucleus pulposus cells in vitro.To observe the transfection efficiency of lentiviral by fluorescence microscopyeasily,the Sox9 gene was labeled with red fluorescence,and the blue fluorescent labeled Fas gene.The experiment was divided into blank control(group(1)),negative control(group(2)),Sox9 over expression(group(3)),Fas interference(group(4)),Sox9 over expression combining with Fas interference(group(5)).The lentiviral vector carrying the target gene was transfected into human degenerative nucleus pulposus cells for 72 hours,and the transfection efficiency was detected by fluorescence microscopy.CCK8 was used to detect the proliferation of cells in each group and RT-PCR was used to test the expression of Sox9,Fas,type II collagen and proteoglycan in m RNA level.The expression of type II collagen and proteoglycan inprotein level were detected using Western-Blot.Results After successful resuscitation of human degenerative nucleus pulposus cells,the cells growed normally and the condition was good.The recombinant lentiviral vector of Fas-si RNA was successfully constructed by enzyme digestion and sequencing of recombinant clones,and the titer was more than 1x108TU/ml.After transfection,the transfection efficiency of the transfected human degenerative nucleus pulposus cells was more than 80% after 72 h.The results of CCK8 showed that there was no significant difference in cell proliferation between the groups(1)and group(2)(P>0.05).The group(3),group(4)and group(5)compared with group(1)in the cell proliferation rate were significantly increased,in which the cell proliferation rate of group(5)increased significantly,the difference was statistically significant(P<0.05).RT-PCR method was used to determine the expression of Sox9,Fas,type II collagen and proteoglycan in m RNA level ingroup(1)was not significantly different compared with the group(2),the difference was not statistically significant(P>0.05).In group(3),Sox9 expression increased significantly in m RNA level,the expression of Fas in group(4)decreased significantly,the expressionof Sox9 in m RNA level in group(5)increased most significantly,Fasexpression was reduced significantly,compared with group(1)the difference was statistically significant(P<0.05).The results of Western-blot showed that the expression of type II collagen and proteoglycan in group(2)was not significant compared with group(1),the difference was not statistically significant(P>0.05).The expression of type II collagen and proteoglycan in group(3)and group(4)were increased ignificantly,group(5)was the most significant.Compared with group(1),the difference was statistically significant(P<0.05).Conclusion Lentivirus mediated Fas-si RNA can significantly inhibit the expression of Fas gene in human degenerative nucleus pulposus cells.Up-regulation of Sox9 gene expression and inhibition of Fas gene expression can increase the expression of extracellular matrix type II collagen and proteoglycan.The experimental results showed that Sox9 and Fas gene were associated with intervertebral disc degeneration,which may prevent or reverse the degeneration of intervertebral disc.