The Effect Of Bmscs On CXCL10/CXCR3 Chemokines Axis And RANKL/OPG Pathways In CIA Rats

Objective:Investigate the effect of BMSCs on CXCL-10/CXCR3 chemokines axis and RANKL/OPG pathways in CIA rats, and to explore the role of MSCs in process of immune regulation mechanism in RA.Method:1.Using freund’s complete adjuvant and type II collagen emulsion immune the Wistar rats twice, then set up the the CIA model. The model observation target including the symptoms of rats(general view, arthritis index,joint swelling degree), imaging and pathological evaluation of joint.2.Using density gradient centrifugation combined with adherent culture method isolation and culture of rat bone marrow mesenchymal stem cells in vitro;Flow cytometry detects BMSCs’ surface antigen CD44、CD105、CD29、CD34、CD45 and CD31 expression,through the exclusion of the cultured cells for identification of BMSCs. Mesenchymal stem cells induced to differentiate into bone cells and adipocytes.3.Grouping: control group(8), CIA model group(8), BMSCs 3d group(8), MSCs30/42 group(8). Transplantation BMSCs in BMSCs groups via caudal vein, cells number of each rat is 1 x 107 / kg.The control group give the same amount of saline in the same way.4.Using Elisa to detect serum RANKL,OPG and CXCL10 level;Separate the spleen, lymph nodes, synovial of each rats, using immunohistochemical to detect CXCR3, TRAF6 protein expression level in those tissues, using RT-PCR to detectCXCL-10/CXCR3, RANKL/OPG, TRAF6 mRNA expression level in those tissues.According to the data distribution using t test or Wilcoxon test to compare each group.Results:1. Bone marrow mesenchymal stem cell were separated from Witar rat successfully,which has the morphological features. Phenotypic characterization expression of CD44, CD105 and CD29, not expressing CD45, CD34, CD31;2. BMSCs transplantation can improve the arthritis index, the degree of joint swelling, X-ray changes and pathological changes of CIA rats;3. BMSCs transplantation can inhibit the CXCL10/CXCR3 chemokines axis of CIA rats, characterized by(1)reduce serum CXCL10 level;(2)inhibit the expression of receptor CXCR3 protein of lymph nodes, synovial tissue;(3) reduce the expression of CXCL10 mRNA of spleen, lymph nodes, synovial tissue, and CXCR3 mRNA expression of lymph nodes, synovial tissue;4. BMSCs transplantation can inhibit the RANKL/OPG pathway of CIA rats,characterized by(1)reduce serum RANKL level and RANKL/OPG ratio;(2)inhibit the expression of intracellular signal transduction pathways factor TRAF6 protein of spleen, lymph nodes, synovial tissue;(3) reduce the proportion of RANKL/OPG mRNA of lymph nodes, synovial tissue, and reduce CXCR3 mRNA expression of spleen,lymph nodes, synovial tissue.Conclusion:1.There exists CXCL10 / CXCR3 chemokines shaft disorder in the process of CIA rats formation, to reduce the CXCL10/CXCR3 level may contribute to the mechanism of BMSCs,which could ease the CIA rats arthritis symptoms.2.There also exists RANKL/OPG ratio imbalance in the process of CIA rats formation. The elevatory RANKL/OPG ratio level give riseto the high expression of receptors in intracellular(TRAF6 protein) resulting in intracellular signal transductionthat mediated bone destruction process; Inhibition of RANKL/OPG bone destruction pathway is the mechanism of BMSCs,which could ease the CIA rats joint destruction.