The Effect Of HTRAIL In The Apoptosis Of Rheumatoid Arthritis Synovial Fibroblasts

Background:The pathology of rheumatoid arthritis is mainly characterized by theproliferation of synoviocytes and the infiltration of inflammatory cells,leading tosynovial hyperplasia and destruction of the cartilage and bone in joints.More and moreresearch indicates that insufficient apoptosis of the rheumatoid arthritis synovialfibroblasts is one of its important pathogenesis.Therefore inducing the apoptosis ofRASFs and inhibiting the hyperplasia of synovium bocome the critical of RAtherapy.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is anew apoptosis inducing ligand which has been found recently.TRAIL can selectivelyinduce the apoptosis of many tumor cells,however it does not adversely affect normalcells.There is one study undertaken to evaluate the ability of adenoviral-mediated genetransfer of human TRAIL to eliminate the hyperplastic synovium in a rabbit model ofIL-1β-induced arthritis,The results demonstrate that the TRAIL mediated apoptosis ofinflammatory synovial is raise up.This study is plan to induce the apoptosis of RASFswith recombinant human TRAIL protein in vitro,hope to look for a new therapeuticapproach of RA.Objective:To investigate the effect of hTRAIL protein in the apootosis of rheumatoidarthritis synovial fibroblasts(RASFs）.Methods:1. Cell culture:Primary synoviocytes were isolated from RA synovium by enzymedigestion.2. The identification of synoviocytes:When the synoviocytes were tranfered to thefourth passage,observed the morphogy with inverted microscope and identificatedthe type II collagen on synovial fibroblasts.3. Cell proliferation:Different concentration of the drugs were added to the basicDMEM, after keep culturing respectively for different time,detected its inhibition toRASFs by MTT assay.4. Flow cytometer（FCM）:The cells were divided into experimental group(basic highglucose DMEM media added with different concentrations of hTRAIL）and control group(basic high glucose DMEM media only),after4h the cells were collected andthen tested the apoptosis of RASFs by FCM.Each assay was performed3times andthe mean data are available.5. RT-PCR:The cells were divided into experimental group(basic high glucose DMEMmedia added with different concentrations of hTRAIL）and control group(basichigh glucose DMEM media only),after4h the RASFs were collected,andimmediately did the RT-PCR reaction.photographed and analyzed by imageanalysis software.Each assay was performed3times.6. Western bloting:DMEM media added with different concentrations of hTRAIL）after4h the cells were collected,did the Western bloting photographed and analyzedby image analysis software.Each assay was performed3times and the mean dataare available.Results:1. Growth inhibition of RASFs by hTRAIL:Treating the synoviocytes with differentconcentrations of hTRAIK for different time,the results indicated that comparingwith control group,the difference of each group had marked significance(P<0.01).2. The RASFs are sensitive to hTRAIL mediated apoptosis.3. Comparing with control group,the difference of DR5mRNA and protein expressionmarked significantly(both P<0.01).4. Comparing with control group,the difference of DR5protein expression markedsignificantly(both P<0.01).Conclusion: hTRAIL can induce the apoptosis of RASFs in vitro.