A Comparative Study Of The Expression Of OPN And NF-kappa B Pathway Proteins In The Synovial Cells Of The Knee Joint

Objective:Osteoarthritis is a chronic joint disease characterized by degeneration of articular cartilage and hyperplasia of articular cartilage and synovial.The previous investigations reveal that the expression of osteopontin(osteopontin,OPN)in synovial tissue of osteoarthritis(osteoarthritis,OA)increased,but the mechanism is unknown.On the other hand,the studies have shown that intracellular NF-kappa B pathway is activated in the articular cartilage,synovial fluid and synovial medium tissue of OA patients,resulting in the corresponding intracellular effects,affecting the progress of osteoarthritis.The purpose of this cases-control study is to investigate the expression of related proteins about Nuclear factor kappa B(NF-KB)signaling pathway and the osteopontin in fibroblast-like synoviocytes of human with knee osteoarthritis.To conjecture the pathogenesis of disease and throw out a new treatment of osteoarthrosis.Methods:1.The study was performed on 15 specimens of synovium obtained from injury of joint and 20 cases from osteoarthritic patients.A total of 20 cases of primary knee osteoarthritis synovial specimens,including 8 cases of male and 12 cases female,average67.2 + 8.5 years old.15 specimens were selected for the control group within 3 days of surgery in patients with synovial specimens of 15 cases of emergency surgery and meniscus or ligament injury in knee fractures,9 cases were male,6 female with an average age of 34.5 + 7.8 years.Comparison of histopathological changes in two groups of synovium by HE staining;Immunohistochemistry and immunofluorescence were used to observe the expression of OPN and NF-kappa B pathway related proteins,inclued p65?I?B?and p-p65 in the synovial tissue of the knee joint of OA patients.2.The enzymatic digestion in normal synovial tissues,isolation and culture of group intervention the first generation of synovial cells,according to the sequence of siRNA synthesis of OPN-siRNA reported in the literature,as a negative control with no significant Con-siRNA.They were divided into 1: normal synovial cells +OPN-siRNA,2normal synovial cells + Lipofectamine 2000,3 OA synovial cells + OPN-siRNA,4 OA synovial cells + Lipofectamine 2000.Morphological observation of the cultured fourth generation fibroblast synoviocytes,Then Western blotting(Western blot)detection of knee joint fibroblast OPN and NF-k the B pathway in p65 and phosphorylation of p65 protein beta-actin used as a reference.3.The positive expression of OPN and NF-kappa B related proteins in two groupsof synovium tissues in two groups of synovial tissues measured by immuno histochemical method in the same multiple field of vision were measured with the software of image analysis.The Image J software was used to analyze the gray value of each protein band in the synovial intervention experiment.Homogenization of the gray value of NF-kappa B pathway related proteins,divided by the average gray level of beta-actin,then the relative quantitative values of each protein are obtained.The data were processed by SPSS19.0statistical software.According to the normal distribution and homogeneity of variance,independent samples t test or Wilcoxon rank sum test were performed.P < 0.05 indicates that the difference is statistically significant,and the influence of OPN and NF-?B signal pathway on the synovitis hyperplasia of knee joint is analyzed.Results:HE staining show that the inflammatory cells infiltration and hyperplasia in group OA;immunohistochemistry and immunofluorescence showed that the expression of OPN and NF-kappa B signaling protein in the pathological specimens of OA patients was significantly higher than that of normal people;The Western blot showed that the expression of OPN and cell pathway protein p65 and P-p65 in OA group was higher than that of normal synoviocytes,and the p65 and p-p65 pathway proteins in the fibrous synovial cells were reduced by the intervened OPN-siRNA experiment than in the control group,The difference was statistically significant.Conclusion:The fibrous synovial cells of the knee joint of OA patients excessively express the OPN,activate the NF-kappa B signal pathway,and produce the downstream effect,which affects the development of osteoarthritis.This may be an important part of the mechanism of synovitis affecting the pathogenesis of OA.