In Vitro Study Of Synovial Membrance Fibroblast-like Cell Infected By RAAV-hOPG

Rheumatoid Arthritis(RA) is a ploygenic,autoimmunity disease with arthritis as its main character.Etiology of RA isn't very clear till now,but we already have a exact insight about the course of immunity and inflammatory reaction.The inflammatory of synovial membrance not only cause pain and swelling of joints,but also irreversible damage of bone and articulus.Osteoprotegerin(OPG) is a protein of 401 amino acids synthesized as a monomer of approximately 55 kDa.OPG belongs to the superfamily of TNF receptor-like proteins,OPGL appears to be an osteoclast differentiation an activation factor.It has been demonstrated that OPG can depress osteoclast differentiation,muture osteoclast activation and induce its apoptosis to incrsease bone mass systematically or locally.Mices who had OPG gene been knockouted show severe osteoporosis after grown up.Mice with ovariectomy shows Osteoporosis.2 weeks after these mices been injected recombinnation human OPG,bone mineral density and bone mass both increased significiantly.The competetive combination between OPG and OPGL(Osteoprotegerin ligand) is thought to be the most important regulatory mechanism in the osteoclast defferentiation,generation,opoptosis and in the course of physiological activation.It suggests OPG has widly application in the treatment of osteoporosis,osseous metastasis, hypercalcemia and Paget's Diseaes.Current studies focus on gene therapy,and the technique of gene transfer plays a key role in successful gene therapy.AAV is a type of DNA virus belongs to Parvoviridae,which has no pathogenicity to human.AAV vector has lots of superiority,and it can effective infect kinds of cells,including divisive and nondivisive cells.All these make AAV vector a new hot point of gene therapy.In this study,we have established AAV mediated gene transfer system and demonstrated that rAAV-hOPG can depress osteoclast defferiation and activity of bone resorption.The study also study the time limit of OPG secretion in the synovial membrance fibroblast-like cell after infected by rAAV-hOPG and if the virus influence cell growth.Partâ… Estanblishment of the rAAV-hOPG and titre determination Objective:estanblishment of the rAAV-hOPG.Methods:estanblish recombination pSNAV2.0-hOPG plasmid,then use it transfects BHK-21 cell,adenovirus,rAAV-hOPG were prepared by series of procedures of picking plaques,amplification,purification and identification.Results:Prepared rAAV-hOPG with high titre(10¹¹pfu/ml).Conlusions: The preperation of rAAV-hOPG(with high titre) was simple and reliable.Partâ…¡Biological Activity Study of synovial membrance fibroblast-like cell after infected by rAAV-hOPG in vitro.Objective:estanblish rAAV-hOPG can infect synovial membrance fibroblast-like cell in vitro,and study its influence to cell growth.Methods:Synovial membrance fibroblast-like cells were cultured in 6 pore plate with DMEM including 10%fetal calf serum(FCS).Move off the culture medium when the Synovial membrance fibroblast-like cells growing to 80%,added 1ml virus fluid which diluted by culture medium (cell:virus=1:10) and keeped culturing.Tested genetic transcrption levels through and OPG expression through Western blot and ELISA.Results:RealTime-PCR showed that the level of OPG genetic transcrption was increased after infected;Western blot and ELISA demonstrated OPG been secrted by the Synovial membrance fibroblast-like cells,and can lasted 8 weeks.There was no difference in cell cycle and growth condition between infected and noninfected Synovial membrance fibroblast-like cells.Conclusions: rAAV-hOPG can efficiently infect Synovial membrance fibroblast-like cells.The cells aider infected can express OPG in a certein term.Partâ…¢Induction of osteoclast and bone resorption testObjective:Investigate the influence of rAAV-hOPG to osteoclast form and function. Methods:Induced mesenchymal(MES) cell to osteoclast by 1α,25(OH)₂D3 and Dexamethasone.Added combination AAV to cultured MES cell in vitro,then did TRAP stain,counted the number of osteoclast and resorption lacune.Results:the number of osteoclast and resorption lacune was significant deduced(p＜0,01).Conclusions: rAAV-hOPG can depress osteoclast differention and activity of bone resorption.