Research On The Correlation Between Newly Discovered OA-related Gene MCF2L Mutation With Apoptosis And Inflammation Of Synovial Fibroblasts

Objective:To discuss the influence of single nucleotide polymorphism(SNP) of a newly discovered Osteoarthritis (OA)-related gene (Mcf.2transforming sequence-like, MCF2L) on the ability of synovial fibroblasts in secreting apoptosis and inflammatory cytokines, These results would further provide clues for the pathological roles of MCF2L in OA.Methods:Bioinformatics were used in collecting and, analyzing MCF2L mutant database and predicting1-2functional Single Nucleotide Polymorphisms (SNP) mutation points potentially important for its function for further study; MCF2L full length gene was obtained from mouse primary synovial fibroblasts, then this gene was overexpressed in adenovirus vector to construct MCF2L overexpressed adenovirus, maeanwhile MCF2L mutant adenoviruses was also constructed according to published MCF2L mutant, then the two kinds of adenovirus were taken for transfection of mouse primary synovial fibroblasts, the expression of MCF2L protein in transfected cells were detected by western blot analysis, Enzyme-linked immunosorbent method test (ELISA) was taken to detect the affection of their influence on OAsynovial cells in secreting IL-1β、matrix metalloproteinase-13(MMP-13)、 Prostaglandin E2(PGE2), cell proliferation was supervised by MTT Assay, flow cytometry(FCM) was conducted to detect cell apoptosis. Results:①Through bioinformatic analysis,We determined the R11L mutant as the target SNP mutant for the follow-up study;②Primary synovial fibroblasts cell was obtained;③Murine MCF2full length gene was successfully obtained and verified;④Overexpressed and mutant adenoviruses were separately constructed in synovial fibroblasts;⑤ELISA test results showed that the expression levels of inflammatory cytokines such as IL-1β ((164.72±20.43pg/ml, P<0.05), MMP-13(103.85±21.03pg/ml, P<0.05) and PGE2(96.07±25.14pg/ml, P<0.05) in MCF2L mutant adenovirus infected fibroblasts were higher than that of over-expressed cell group, MTT showed that the MCF2Lmutant cells inhibited the proliferation of synovial fibroblasts, flow cytometry showed that, in MGF2L mutant synovial fibroblasts, the expression level of IL-6, TNF-a and other pro-apoptotic factors were higher than the over-expressing cell group,which were in consistent with inflammatory factors.Conclusion:The recombinant adenosvirus with the MCF2L (Rl1L) mutant infected the synovial fibroblasts that induced inflammation and degenerative pathological changes in the knee joint. The increase expression of inflammatory factors in the serum of IL-1β, MMP-13and PGE2explained the OA. This can helpful for further understanding of the mechanism which engages in knee osteoarthritis and provide the references for development of new prevention and control measures and support the MCF2L as diagnose gene for clinical usage.