In Vivo Experimental Study Of Lentiviral Mediated MMP3-siRNA And Sox9 Transfer Degeneration Nucleus Pulposus In Rabbits

Objective: To develop an animal model of intervertebral disc degeneration and use MMP3-siRNA and Sox9 Lentiviral Vector(LV)to tranfect and explore the biological effects of single gene and double genes transfection of rabbit disc degeneration.Methods:An animal model of disc degeneration was developed with acupuncturing annulus fibrosus in rabbit(average 4 moths old),then the MRI and histopathology were performed after operation 4 weeks and 8 weeks,which aimed to observe the success rate of making intervertebral disc model.30 New Zealand white rabbits on the basis of successful modeling were devided into 6 groups: Group 1: Blank control;Group 2:Phosphate buffer saline;Group 3: rLVX-mCMV-mcherry;Group 4: rLVX-CMVhMMP3-shRNA-mCMV-mcherry;Group 5: rLVX-CMV-hSox9-m CMV-mcherry;Group 6: rLVX-CMV-h MMP3-shRNA-mCMV-mcherry + rLVX-CMV-hSox9-mCMV-mcherry,with 20 ul injection respectively.Subsequently,the MRI was performed after injection 8,12,24 weeks to observe the degeneration of intervertebral disc.The animal model were sacrificed at 24 weeks after operation,then Western-blot analysis of the intervertebral disc tissue was for the expression of Collagen Type II and proteoglycan,RT-PCR analysis was for the expression of MMP3-siRNA and Sox9 RNA.Results: The MRI T2 WI after operation 4 and 8 weeks showed that the signal intensity of T2 WI was weaken in the group 8 weeks,and the histopathology showed the intervertebral disc was degeneration,which indicated the models were established successfully.The MRI was performed after transfection 8,12 and 24 weeks with target gene,the result showed that the signal intensity of T2 WI in group Blank control,PBS and the empty vector control group was lower than MMP3,Sox9 and double genes group.Comparition between the signal of T2 WI of group Blank control,PBS and the empty vertor control group had no statistics significance(P>0.05).There was no statistics significance between the T2 WI of MMP3 and Sox9(P>0.05).There was significance difference between the MRI of MMP3,Sox9,and double genes through respectively comparison(P<0.05).In the expression of Collagen Type II and proteoglycan after the operation 24 weeks,Collagen Type II of the MMP3,Sox9 and double genes was increased,and double genes group was higher than other groups.When compared the expression of proteoglycan(P<0.05),MMP3 and double gene group were higher(P<0.05).There had no significant increase in the Sox9 group(P>0.05).The expression of double genes group was higher than other groups(P<0.05).In the expression of mRNA between 5 groups,MMP3 group had a significant decrease(P<0.05),and double genes group was lower than others(P<0.05).There was a significant increase in the expression of Sox9 mRNA(P<0.05).Moreover,the expression of Sox9 in double genes group was higher than other groups(P<0.05).Conclusions: This study performed the development of disc degeneration model with acupuncturing annulus fibrosus,and the imaging and histopathology indicated the model was efficient.The degenerative nucleus pulposus cells of rabbits transfected by Lentiviral Vector mediated MMP3-si RNA and Sox9 can delay the degeneration of intervertebral disc effectively by promoting the expression of Collagen Type II in the degenerative disc,and the transfection of MMP3-siRNA can increase the expression of proteoglycan while the overexpression of Sox9 can't promote the expression of proteoglycan.