Study On Autophagy In Heterotopic Ossification Of Ankylosing Spondylitis Peripheral Joint And In Necrosis Of Femoral Head

The first section Study on the mechanism of autophagy in heterotopic ossification of ankylosing spondylitis peripheral jointPURPOSE:1.Whether LC3Ⅰ/Ⅱ,Beclinl and P62,markers of autophagy elevate in synovial tissue and synovial fibroblasts from ankylosing spondylitis patients when Comparing with synovial tissue and synovial fibroblasts from popliteal cyst respectively;2.Whether the osteogenic function of synovial fibroblasts from ankylosing spondylitis patients is regulated by autophagy of their own.METHOD:1.Experimental designing and grouping Grouping:synovial tissue from from ankylosing spondylitis patients,from popliteal cyst.synovial fibroblasts from ankylosing spondylitis patients,from popliteal cyst and synovial fibroblasts from ankylosing spondylitis patients co-cultured with drug which can regulate autophagy.The expression of ALP,LC3Ⅰ/Ⅱ,Beclinl and P62 will be detected in different groups.2.Experimental content2.1 Detecting ALP,LC3Ⅰ/Ⅱ,Beclinl,P62 in the synovial tissue with immunohistochemisty.2.2 Synovial tissue is digested with collagenase(0.2%type Ⅰ collagenase)to get synovial fibroblasts.2.3 The surface markers CD55,CD90,CD14 are detected with flow cytometry to verify the purity of synovial fibroblasts.The MTT assay is used to calculate the proliferation of synovial fibroblasts from different groups,and detect the toxicity of drug which can regulate autophagy to synovial fibroblasts from ankylosing spondylitis patients.2.5ALP,LC3Ⅰ/Ⅱ,Beclinl and P62 of synovial fibroblasts from each group are detected with Western blotting.3.Statistical analysisSPSS 20.0 statistical software is used to analyze the data,and one-way ANOVA to test the experimental data.P<0.05 is set to a statistically significant difference.RESULT:1.The expression of ALP,LC3 and Beclinl in synovial tissue from ankylosing spondylitis patients is significantly higher than that from popliteal cyst(P<0.05).The expression of P62 is opposite,but there is no statistically significant difference between groups.2.There are no significant differences in the ratio of synovial fibroblasts between different groups,and all are more than 95%.3.Proliferative capacity from high to low is synovial fibroblasts from ankylosing spondylitis patients,from ankylosing spondylitis patients co-cultured with 3-methyladenine and from popliteal cyst.There is statistically significant difference among groups.Without significant cytotoxicity to synovial fibroblasts,the maximum concentration of 3-methyladenine is 7mmol/1.4.The ratio of LC3Ⅱ and LC3Ⅰ and the expression of ALP,LC3Ⅱ,Beclinl from high to low are synovial fibroblasts from ankylosing spondylitis,from ankylosing spondylitis co-cultured with 3-methyladenine and from popliteal cyst.There is statistically significant difference between the former and the two latter.Over time,the ratio of LC3Ⅱ and LC3Ⅰ and the expression of ALP,LC3Ⅱ,Beclinl synovial fibroblasts from ankylosing spondylitis co-cultured with 3-methyladenine decreases,and there is significant difference among groups co-cultured with 3-methyladenine.Although the expression of P62 is different,no significant difference is found among groups.CONCLUSION:1.Increased autophagy level in synovial tissue and synovial fibroblasts from ankylosing spondylitis patiens.2.Autophagy can positively regulate heterotopic ossification in the soft tissue around joints of ankylosing spondylitis patients.The second section Study on the correlation between autophagy and synovial lesions of necrosis of femoral headPURPOSE:The expression levels of LC3,Beclinl and P62 protein in the synovium from PC and ONFH are detected to determine whether the synovial lesion of ONFH is associated with autophagy.METHOD:The expression levels of LC3,Beclinl and P62 protein in synovium from 11 cases of PC and 11 cases of ONFH(no clear etiology,ARCO Ⅲ stage and above)are detected by immunohistochemistry.RESULT:The expression of LC3 and Beclin-1 in the synovium from ONFH is higher than that from PC,but there is no significant difference between groups.The expression of P62 is almost none in the two groups.CONCLUSION:There is no correlation between synovial lesion and autophagy in ONFH(ARCO III and above).