| Osteoarthritis (OA) is the most frequent arthropathy among middle-aged people. Prevalence studie show that OA usually increases with ages, and it affects more than 10% of the population older than 45 years and up to 50% over the 60s. The knee is the most commonly affected by osteoarthritis, which mainly occurs in the more weight bearing and activities joints. The major clinical manifestations of osteoarthritis are joint pain, tenderness, stiffness, swelling, bone fricative, joint weakness, movement disorder and etc. Studies have shown that disability rate is high up to 53%, and more than 80% of all patients reported limitations in their activities of daily living, either for basic tasks, leisure activities, or work. Some are primary osteoarthritis, the others are secondary osteoarthritis, the later of which because of accumulating knee damage, such as injury, inflammation, joint instability, chronic repeated injury. The etiology of primary osteoarthritis is unknown. A large number of studies have shown that primary osteoarthritis is related to age, obesity, genetics, immune responses, biomechanical changes, the metabolic balance of chondrocytes and matrix. In recent years, with the rapid development of molecular biology and molecular immunology, more and more attention are paying to the metabolism media of cartilage and synovial fluid, especially cytokines and inflammatory mediators. In articular cartilage, chondrocytes play an important role in the synthesis and decomposition of extracellular matrix and the stability of joint structure are maintained by the balance of chondrocytes and extracellular matrix. When the microenvironment of articular cavity is changed, the matrix balance of synthesis and decomposition become abnormal, which lead to damage of cartilage. Through the role of mechanical stress, cytokines and so on, OA is characterized by a progressive loss and necrosis of articular cartilage, osteophyte formation, thickening of subchondral bone, and subchondral cyst formation, which lead to the destructure of joint structure at the end.As everyone knows, articular cavity is a hypoxia environment. It has been reported that the partial pressure of O2 in synovial fluid of joints affected by osteoarthritis (OA) is between 50-60mmHg, far less than other tissues. Articular cartilage is an avascular tissue getting its oxygen and nutrients from the dynamic flow of synovial fluid during joint movement or from the subchondral bone. Normal cartilage cells derived 75% ATP from anaerobic metabolism, which survived to the intra-articular hypoxia. Therefor, the metabolism balance of oxygen has an important role in maintaining the stability of articular structure. Most mammalian cells possess an oxygen-sensing system and control oxygen homeostasis by up-regulation of a diverse set of genes including erythropoietin (EPO), vascular endothelial growth factor A (VEGF-A), induciblenitric oxide synthetase (iNOS), glucose transporters, enzymes of the glycolytic pathway and leptin. Among these factors, hypoxia inducible factor 1 (HIF-1) is the most important regulatory in the hypoxic environment.Hypoxia inducible factor 1 (hypoxia-inducible factor, HIF-1) was found in the study period of erythropoietin (erythropoietin, EPO) in hypoxia, which is a DNA binding protein. HIF-1 consists of the subunit HIF-1α(120 kDa), produced in response to hypoxia, and the constitutively expressed HIF-1β(91 to 94 kDa) subunit. It belongs to the family of basic helix-loop-helix(bHLH) containing PER-ARNT-SIM(PAS) domain transcription factors, which is the specific regulation factor of HIF-1 in hypoxia. A large number of studies have shown that HIF-1 plays an important role in cancer, inflammation, ischemic tissue and the development of growth plate. The high expression of HIF-1 aggrandize the expression of erythropoietin (EPO), vascular endothelial growth factor (VEGF-A), inducible nitrogen oxide synthase (iNOS), glucose transporter enzyme, glucose catabolic enzymes and leptin. Early detection found the expression of HIF-1 mRNA in articular cartilage cells in hypoxia, followed the osteoarthritis found also. However, the function of HIF-1 a is not very clear.In recent years, the study in the pathogenesis of osteoarthritis pays more and more attention to synovitis. A large number of studies have shown that synovitis has an important role in the pathogenesis of osteoarthritis. Synovitis in advanced OA may be as severe as in rheumatoid arthritis (RA). While a large number of studies in terms of the arthroscopy, Magnatic Resonance Imaging (MRI), ultrasound and molecular biology demonstrated the role of synovitis in development process of osteoarthritis. When the synovial proliferation and degeneration of articular cartilage go ahead, the no degeneration cells are more difficult to resist harsh environments which further intensified of the hypoxia. But studies about the expression of HIF-1αin synovium and the cell resistance to hypoxia are rarely reported.In this study, the expressions of HIF-1αwere detected by immuneohis-tochemical staining in different knee diseases and normal control group, in order to understand the difference expression of HIF-1αbetween the diseases, and its relationships with downstream protein. Further investigate its possible role in the progress of osteoarthritis. We extracted HIF-1mRNA from human synovium, and constructed overexpressed vector of HIF-1 a with gene transfer technology. The overexpression vectors and siRNA HIF-1αvector which we purchased were transfected into normal synovial cells (HS-4700). In order to understand the function of HIF-1αin transgenic cell under hypoxia, we detected the cell morphology and cell proliferation in plate clone experiment in hypoxia.Chapter one:Expression of HIF-1αin human synovial tissueObjective:To detect the expression of Hypoxia-inducible factor-1α(HIF-lα), Vascular endothelial growth factor(VEGF) and CD34 in normal human synovium, patients with acute traumatic arthritis, chronic non-specific synovitis and osteoarthritis. Analysis the relevant relationships and investigate their relationships in the pathogenesis of osteoarthritis.Methods:The expressions of HIF-la and VEGF were detected by immunohistochemical staining in 30 patients with osteoarthritis,25 with acute traumatic arthritis,45 with chronic non-specific synovitis and 10 normal synovial biopsy. CD34 staining was produced on the synovium of chronic non-specific synovitis and normal control group. The correlation and differences between HIF-la, VEGF, capillary density (MVD) and their relationships to the diseases were analyzed.Results:There were almost no HIF-1a and VEGF positive expressions in normal human synovial. The positive rates of HIF-1a were respectively 60% in acute traumatic synovitis,86.7% in osteoarthritis, and 68.9% in chronic non-specific synovitis. The positive rates of VEGF were respectively 48% in acute traumatic synovitis,80% in osteoarthritis, and 80% in chronic non-specific synovitis. The positive rate of HIF-la and VEGF were no significant differences in osteoarthritis group and the chronic non-specific synovitis (p> 0.05). But both higher than acute traumatic synovitis group, the difference was significant (p=0.000). The expressions of HIF-la were positively correlated with the expressions of VEGF in three synovitis group, correlation coefficients were respectively 0.596 in acute traumatic synovitis, 0.525 in chronic non-specific synovitis, and 0.600 in osteoarthritis. MVD (CD34 staining positive) was higher in chronic synovitis group than normal, and HIF-la positive specimens were higher than the negative.Conclusion:The expression of HIF-la and VEGF in synovial tissue of patients with osteoarthritis was significantly increased, point out that HIF-la may have an important role in the process of joint degeneration; there were high expression of HIF-la and significantly positive correlated with VEGF in degenerative, inflammation, and trauma synovial tissue, indicate that HIF-la promotes synovial proliferation in the development process of the synovitis; MVD expression in the HIF-la positive synovial was higher than the negative, and HIF-la has an important role in angiogenesis.Chapter Two:the function and morphology of human synovial cell (HS-4700 cell) after HIF-1a transfectionObjective:To establish the overexpression and gene knockout HIF-la plasmid and transfect in normal synovial cell, and to understand the function and morphology of cell after transfection in hypoxic condition.Methods:①To understand gene expression of HIF-la and VEGF in different group, we used RT-PCR to extract HIF-1amRNA, VEGF-AmRNA in Synovial tissue, which were obtained at surgery in patients with osteoarthritis, acute traumatic synovitis, chronic non-specific synovitis and control group.②ccording to the well-known principle of corresponding DNA template design, cDNA were designed and selected by reverse transcription. Then oligos were inserted into pcDNA3.0 plasmid. The HIF-1a/pcDNA3.0 vectors were verified by sequencing.③The HIF-1a/pcDNA3.0, empty vector of pcDNA3.0 and the HIF-siRNA plasmid vector plasmid were transfected into human normal synovial cell (HS-4700). When the cells were incubated for 4 hours in the hypoxia incubator (1% oxygen,5% carbon dioxide, 94% nitrogen), cell morphology was observed under inverted microscope and Western Blot assay were done in different synovial cell group.④To understand the effects of HIF-la on cell proliferation, we done the cell plate cell proliferation on transfected synovial cells, which were cultured in medium containing CoCl2.Results:①RT-PCR results showed that gene expression of HIF-la and VEGF was significantly increased in synovial tissue with osteoarthritis. The expression of HIF-la and VEGF mRNA increased in acute traumatic synovitis and chronic non-specific synovitis group too.②The HIF-la/pcDNA3.0 vector was successfully constructed, because the DNA sequencing was confirmed with the same DNA sequence of HIF-1a which designed in the exactly experimentation.③After hypoxia, the group of overexpression HIF-1αwas no obvious morphological changes, and HIF-1αprotein significantly increased. The cell of empty vector group was mild shrink, and expression of HIF-1αprotein increased slightly. The group of siRNAHIF-1αwas significantly shrunk, and no HIF-1αprotein was detected.④Through the plate cloning experiment in hypoxia, many cell clones were detected in the group of HS/HIF-1α. After contrasted the clones by one-way ANOVA, the cell proliferation was significantly increased in HS/HIF-1αgroup than the other two groups (F= 195.852, P=0.000).Conclusion:①The gene expression of HIF-1αand VEGF are similar with protein expression, which are significantly increased in synovium of osteoarthritis;②the expression of HIF-1αin synovial cells are mild in normal oxygen, but increased significantly under hypoxia;③HIF-1αgene has a protective effect on cell proliferation and morphology under hypoxia for cells, which can change the way of metabolism of synovial cell and adapt to harsh hypoxic environment.④HIF-1αcan promote the function of cell proliferation under hypoxia, and play an important role in the phase of synovitis proliferation. |
