The Effect Of P27 Gene On Mice IDD Mediated By Sonic Hedgehog (Shh) Signaling Pathways

Chapter 1The establishment of a new kind of the experimental model for intervertebral disc degeneration in tail suspension miceObjective To establish an easy operation,facilitate,and useful mice intervertebral disc degeneration model.Methods Seventy four wild type(WT)4-week-old SPF mice were randomly divided into two groups:Control group(Con),and Tail Suspension group(TS).The computer X-ray,hematoxylin-eosin(HE)staining and Western-blot were carried out in three time points:2 weeks,4 weeks and 8 weeks.The intervertebral disc degeneration was evaluate by intervertebral disc height index(DHI)through analyzing the mice lumbar lateral scan X-Ray photos,the changes of mice intervertebral nucleus pulposus and annulus fibrosus were observed by HE staining,and the p27 protein was detected by Western-blot in intervertebral disc tissue.Results The X-ray photos demonstrated that the DHI of TS mice was a trend of gradual decline from 4 weeks and the structure of centrum was instable.In week 8,the DHI further decreased,the scoliosis was appeared,and the calcification of centrum edge and cartilaginous endplate was observed intension.However,the Con group showed slightly narrow of DHI in 8 weeks.The differences between the tail suspension group and control group were significant(P<0.05).The HE staining results showed that intervertebral disc nucleus pulposus cells decreased and the stromal collagen fibers arrangement was disorder in TS group,which indicated that serious degeneration occurred in nucleus pulposus and annulus fibrosus.However,the collagen fibers arranged neatly and intervertebral disc degeneration was mild in control group.The western-blotting proved that the level of P27 protein was significantly higher in TS group than in control group(P<0.05).Conclusion The mice intervertebral disc degeneration model was established successfully using the 8 weeks mice tail suspension method,which showed the advantages of nondestructive,easy operation,stability,and reproducible.Chapter 2 The effect of p27 gene in mice intervertebral disc degenerationObjective To identify the role of p27 gene in mice intervertebral disc degeneration(IDD).Methods Four-week-old WT and p27 knockout(p27-/-)mice IDD were produced using the method mentioned in Chapter 1.The mice without tail suspension were chosen as control.The mice were divided into four groups:Control-Mild Type(Con-WT),Control-p27 knock out(Con-p27-/-),Tail Suspension-Wild Type(TS-WT)and Tail Suspension-p27 knock out(TS-p27-/-).The intervertebral disc height index(DHI)was calculated according to the lumbar X-ray images by radiography that could clarify the degeneration of lumbar and intervertebral disc.The lumbar intervertebral disc nucleus pulposus/annulus fibrosus degeneration degree was evaluated by HE staining.Results The lumbar spine X-ray images showed that the lumbar sequence was well-ordered in Con-p27-/-group and Con-WT group mice,no obvious osteophyma formation and DHI narrow.However,the lumbar sequence of TS-WT groups was bone hyperplasia on the edge of vertebral body and the height of disc space was reduced.Moreover,Lumbar scoliosis was exist in some mice.The statistical results show that the DHI of TS-p27-/-is higher than Con-WT groups(P<0.05).HE staining identified that the intervertebral disc fiber and the nuclear pulposus of Con-p27-/-and Con-WT groups was distinct and clear.The nucleus pulposus contained rich cavitation and small chondroid cells and the fibers arranged orderly.Meanwhile,the disc annulus fibers was serious disorder or even big crack and the number of nucleus pulposus cells reduced or even disappeared in TS-WT group,while the disc annulus fibers arrangement was still regular.The intervertebral disc histological score was significantly lower than TS-p27-/-and Con-WT(P<0.05).Conclusion The intervertebral disc degeneration was alleviated when p27 was knockout.P27 gene may play a negative regulation role in the development of IDD.Chapter 3 The effect of p27 gene on mice IDD mediated by Sonic Hedgehog(Shh)signaling pathwaysObjective To clarify whether the negative effect of p27 gene on mice lumbar disc degeneration through Sonic Hedgehog(Shh)signaling pathway.Methods The spine and lumbar intervertebral disc were harvested from mice used in Chapter 2.The related protein levels of Shh signaling pathway in lumbar intervertebral disc were detected by immunofluorescence staining and the expression of these proteins were determined by Western-blotting.The quantification of fluorescence density value and protein band gray value were analyzed by IPP and Photoshop software.Graphpad software was employed to do the statistical analysis.Results The fluorescence and western-blotting results showed that the Shh,Smo,Patched and Gli-2 proteins were detected in all of four groups(Con-WT,Con-p27-/-,TS-WT and TS-p27-/-),among which,Con-p27-/-group has the most highest level of Shh,Smo,Patched and Gli-2 proteins.The four kinds of proteins in TS-p27-/-group was slightly decreased,but still higher than Con-WT group.The lowest protein level group was TS-WT.There were statistical significance difference between each group(P<0.05).Conclusion The p27 gene knockout can active the Shh signaling pathway and slow down the intervertebral disc degeneration which can protect the IDD in mice.