Global Analyses Of Gene Expression In Primary Osteoarthritis Synovium Of The Knee Joint Of Humans

backgroundPrimary osteoarthritis of the knee (KOA) also known as idiopathic osteoarthritis of the knee, and osteoarthritis (OA) is the most common degenerative joint disease in the aged people all over the world. In a previous epidemioIogical study, prevalence of KOA reachs 5%,and it increased to 15-20﹪in the aged people older than 65 years. With the ageing of the population, the prevalence show an upward trend year by year; the etiology, pathogenesy, and effective prevention and treatment of KOA have become an important global subject in orthopaedicics.Generally,the prevalence of primary osteoarthritis of the knee are closely related with age, sex, obesity, genetics, hormones, sports, trauma and occupation.Both mechanical and biological factors lead to normal coupling imbalance of degradation and synthesis in the cartilage cells,extracellular matrix and subchondral bone, articular cartilage damage or degeneration initate the articular pathological changes in the osteoarthritis of the knee. The knee pain, stiffness, limitation of activity and friction noise accompanying the activities are mainly clinical characteristics , and osteoarthritis is one of the most common disabling conditions, affecting many parts of the joint, including bone, synovium, ligaments, and articular carilage.The knee pain, Varus knee or valgus knee, joint space disappeared with flexion deformity will appear gradually in mild and moderate and severe late-stage OA.Diagnosis of OA commonly depends on clinical and radiographic findings. However, changes in cartilage associated with the early stage of OA cannot be detected using radiographs, because significant cartilage degeneration must occur before radiographic findings show alterations of the appearance of cartilage. Synovium is an important part of Synovial joint sliding system with large number of the rich blood supply and nerve supply; the secretion of synovial, absorption and nutrition supply to maintain normal articular cartilage metabolism and function. Study indicates that the synovial cell apoptosis severely increases in osteoarthritis synovium of the knee joint of humans than normal synovium of the knee joint.In the pathogenetic study, much attention has been focused on developing assays for cartilage molecules in the primary osteoarthritis of the knee joint of humans,but this is the first report of the use of whole genome expression profiles chip technology to analyse normal synovium of the knee joint of humans and primary osteoarthritis synovium of the knee joint of patients.ObjectiveDespite many research efforts in recent decades, the major pathogenetic mechanisms of primary osteoarthritis of the knee, including gene alterations occurring during osteoarthritis cartilage degeneration, are poorly understood, and there is no disease-modifying treatment approach. This study was therefore initiated in order to identify all differentially expressed disease-related genes using global analyses of gene expression in primary OA synovium of the knee joint of humans, according to the changes in gene function,and identify potential therapeutic targets to found solid foundation for gene therapy .Some differential expression genes were performed real time quantitative polymerase chain reaction in order to further identify the differential expression genes obtained in primary OA synovium of the knee joint of humans and to find the significance in the process of disease.Methods1.According to addition and exclusion standards, 12 patients with OA were performed , and OA synovium of the knee joint of humans was obtained at the time of arthroscopic surgery and total knee replacement.According to Kellgren and Laurence radiology classification standards 4 patients became a group, then mild(A3)and moderate(A4)and severe late-stage OA groups(A5)appeared. In addition,4 normal synovium of the knee joint samples became B1 group.2.RNA sample preparation(1)two-dimensional separation(2)RNA praecipitatum(3)RNA emundation (4)again dissolve RNA praecipitatum(5)RNA mass detection3.aRNA sample symbol and synthesis(1)cDNA synthesis(2)second-strand of cDNA synthesis(3)aRNA synthesis(4)aRNA depuration(5)aa-UTP aRNA indirect labelling(6)quality control4.chip prehybridization5.Hybridisation6.chip emundation7.result scanning, data reading8.synthetical cDNA were used to real time quantitative polymerase chain reaction9.performing real time quantitative polymerase chain reactionResultsAll the 16 samples were performed the genome-wide analysis of chips, among the 16 samples, the 39,202 genes were expressed in evrey sample, and each sample contained a small amount of different genes compared with other samples.According to the foregoing groups, the 39,202 genes within each group were corresponding calculated the mean, each group was considered as a entirety (A3, A4, A5, B1 four groups), comparing among the four groups. First, we performed a comparison between the disease groups(A3,A4,A5 three groups)and the mormal groups(B1 group)to identify differential expression genes among the three groups in common.In this experiment, among the 39,202 genes, many differential expression genes were identified, and 73 genes showed statistical significance, of which: 46 genes were up-regulation 2-fold, 10 genes were up-regulation 4-fold, 5 genes were up-regulation 8-fold; 27 genes were down-regulation 2-fold, 3 genes were down-regulation 4-fold, 1 genes were down-regulation 8-fold , very much look forward to including anabolism and catabolism matrix gene function change. Particularly important oxidative defense gene were down-regulation,superoxide dismutase 2 and 3,and glutathione peroxidase 3 particularly conspicuous. This showed that successive oxidative stress of osteoarthritis in the cells and the matrix was a major potential pathogenesis.The genes related cellular phenotypic stability significant decreased in primary OA synovium of the knee joint of humans,that indicated those genes were restrained.It is another factor of accelerating degradation of the joint.Second,we performed comparison among the three disease groups to identify differential expression genes in mild and moderate and severe late-stage primary OA synovium of the knee joint of patients,but the results showed 73 differential expression genes have not significant deviation in the disease groups.According to the results of scientific experiments to be reproduceed, 5 differential expression genes were optional selected to perform real time quantitative polymerase chain reaction, through three times repetitive experiments , we miraculously observed the same results of the experiments.Through the statistical treatment, P values less than 0.01 were considered significant to further confirmed differential expression genes in OA synovium of the knee joint of patients played an important role in the process of primary OA.Conclusion1.Differential expression genes in primary OA synovium of the knee joint is the root causes in the development process of primary OA.2.Differential expression genes in primary OA synovium of the knee joint and the function of those genes have no changes following the changes of the development process .3.Both the genes related in anabolism were down-regulation and the genes related in catabolism were up-regulation accelerate the degeneration of synovial knee, thereby speeding up the entire process of degeneration of the knee.4.Phagocytic cell in OA synovium become overacfivity because the genes related immunization were up-regulation, breaking the balance of destruction and renovation, then the knee synovium, cartilage and other structures destroyed gradually.5. Coordination of the synovium of the knee joint metabolism was broken because the the genes related information transfer were down-regulation, thus leading to metabolic disorder of the knee.6.Many genes unite to initiate the Occurrence and development of primary OA of the knee, it also confirmed organization aging theory from another side.