Experimental Study Of Lentivirus-mediated Transfection Of Fas-siRNA And Sox9 Into Rabbit Degenerative Nucleus Pulposus Cells In Vivo

Objective:To transfect rabbit degenerative nucleus pulposus cells by lentivirus-mediated Fas-si RNA and Sox9 in vivo,and to observe the biological effects of single gene transfection and double gene cotransfection on degenerative intervertebral disc.Methods:The healthy adult experimental rabbits were randomly divided into 5 groups: A.PBS group,B.Empty virus group,C.Fas-si RNA group,D.Sox9 group,E.Double gene group;Rabbits models of degenerative intervertebral disc were established by acupuncturing discs,5 groups of rabbit intervertebral discs were injected by using micro syringe with 20 u L PBS phosphate buffer,lentivirus,r LV-h Fas-sh RNA,r LV-h Sox9,r LV-h Fas-sh RNA+r LV-h Sox9 respectively;At the time of 8 weeks,12 weeks and 24 weeks after transfection,the experimental animals were carried on MRI examination respectively to observe the signal changes of intervertebral discs.Then the experimental animals were killed to get the degenerated intervertebral discs.The levels of II collagen and proteoglycan in the intervertebral discs were detected by Western-Blot technology.The expression levels of Fasm RNA and Sox9 m RNA were detected by RT-PCR technology.Results: At the time of 8 weeks,12 weeks and 24 weeks after transfection,the experimental animals were carried on MRI examination respectively to observe the signal changes on T2-weighted images of intervertebral discs.The results showed that:The signal intensities on T2-weighted images of A and B groups were significantly lower than those of C,D and E groups,the differences were statistically significant(P<0.05);The signal intensities on T2-weighted images of A and B groups were about equal,the differences were not statistically significant(P>0.05);The signal intensities on T2-weighted images of E groups were significantly higher than those of C and D groups,the differences were statistically significant(P<0.05).Then the experimental animals were killed to get the degenerated intervertebral discs.The levels of II collagen in the intervertebral discs were detected by Western-Blot technology.The results showed that: The levels of II collagen in the intervertebral discs of C,D and E groups were significantly higher than those of A and B groups,the differences were statistically significant(P<0.05);The levels of II collagen in the intervertebral discs of C and D groups were about equal,the differences were not statistically significant(P>0.05);The levels of II collagen in the intervertebral discs of E groups were significantly higher than those of C and D groups,the differences were statistically significant(P<0.05).The levels of proteoglycan in the intervertebral discs were detected by Western-Blot technology,the results showed that:The levels of proteoglycan in the intervertebral discs of C and E groups were significantly higher than those of A,B and D groups,the differences were statistically significant(P<0.05);The levels of proteoglycan in the intervertebral discs of A,B and D groups were about equal,the differences were not statistically significant(P>0.05);The levels of proteoglycan in the intervertebral discs of E groups were significantly higher than those of C groups,the differences were statistically significant(P<0.05).The expression levels of Fasm RNA and Sox9 m RNA were detected by RT-PCR technology.The results showed that: The expression levels of Fasm RNA of C and E groups were significantly lower than those of A,B and D groups,the differences were statistically significant(P<0.05);The expression levels of Fasm RNA of A,B and D groups were about equal,the differences were not statistically significant(P>0.05);The expression levels of Fasm RNA of E groups were significantly lower than those of C groups,the differences were statistically significant(P<0.05);The expression levels of Sox9 m RNA of C,Dand E groups were significantly higher than those of A and B groups,the differences were statistically significant(P<0.05);The expression levels of Sox9 m RNA of A and B groups were about equal,the differences were not statistically significant(P>0.05);The expression levels of Sox9 m RNA of D groups were significantly higher than those of C groups,the differences were statistically significant(P<0.05);The expression levels of Sox9 m RNA of E groups were significantly higher than those of D groups,the differences were statistically significant(P<0.05).Conclusion: The lentivirus-mediated transfection of Fas-si RNA and Sox9 into rabbit degenerative nucleus pulposus cells in vivo can inhibit the degradation of II collagen and proteoglycan in extracellular matrix,reduce the apoptosis of nucleus pulposus cells,and increase the content of II collagen and proteoglycan,thus postpone the degeneration of intervertebral disc.Double gene cotransfection can postpone the degeneration of intervertebral disc better than single gene transfection.Synergistic effects exist between the two genes.