Study On The Biological Effects Of MMP3 And SOX9 Gene Regulation In Human Intervertebral Disc Degeneration Of Nucleus Pulposus Cells Mediated By Lentiviral

Objective:To detect the Biological effects of MMP-3 and SOX-9 gene regulation in human intervertebral disc degeneration of nucleus pulposus cells mediated by lentiviral.Methods:By the method of sequential enzyme digestion,we separate the pulp tissue from the patients who suffer from intervertebral disc herniation,then we get monolayer primary cell culture(group)from the polp tissue,then we culture the nucleus pulposus cells of human degeneration intervertebral disc and develop(Our teamwork have Cryopreserved cells).We design and get the lentiviral of MMP3-siRNA and SOX9 gene,then we finished the lentiviral packaging.We use lentiviral to transfect nucleus pulposus cells of human intervertebral disc degeneration.Then we observe the cell morphology and cell structure,and identify it by h-e staining.We detect the lentiviral transfection efficacy by different concentration of lentiviral in order to find the concentration that have the highest transfection efficiency.We detect the effect on the proliferation of nucleus pulposus cells of human intervertebral disc degeneration when silence MMP3-siRNA gene and overexpression SOX9 gene.Then we use RT-PCR and Western-Blot to detect the influence of MMP3 gene silencing and SOX9 gene over expression on nucleus pulposus cells of human intervertebral disc degeneration,include the secretion of polysaccharide and the expression of type ? collagen at mRNA and protein level.Results:We found that the original generation nucleus pulposus cells of human intervertebral disc degeneration have the same cellular morphology(triangular,short polygon or fusiform,irregular shape,the nucleus is bigger).Immunofluorescence found that the determination of lentivirus transfection efficacy,the transfection efficiency can reach to80-90%.MTT showed the blank group and MMP3-siRNA/SOX9 lentivirus empty carrier growth was no significant difference on cell proliferation.The group of MMP3-si RNAgene interference and the group of SOX9 gene over expression can promote nucleus pulposus cells of human intervertebral disc degeneration proliferation,and the group both silence MMP3-siRNA gene and overexpress SOX9 gene is more obvious.RT-PCR detection showed: The group of MMP3-siRNA silent can promote the cells secreted polysaccharide and type ? collagen(P<0.05).The group of SOX9 gene overexpress also can promote the cells secrete polysaccharide and type ? collagen(P<0.05).The group that both silence MMP3-si RNA and overexpress SOX9 gene have the strongest ability.Western Blot results show: The group of MMP3-siRNA gene silent can promote the cells secreted polysaccharide and type ? collagen(P<0.05),The group of SOX9 gene overexpress also can promote the cells secreted polysaccharide and type ? collagen(P<0.05),the group that both silence MMP3-siRNA and overexpress SOX9 gene have the strongest ability.Conclusions:When MMP3-siRNA gene silencing and SOX9 gene over expression,it can promote nucleus pulposus cells of human intervertebral disc degeneration proliferation,and can promote it secreted protein,polysaccharide,type ? collagen secretion at mRNA and protein level.In conclusion,MMP3-siRNA gene and SOX9 gene might as new target that can be prevent or reverse cervical disc degeneration.