| ObjectiveOsteoarthritis, also known as degenerative arthritis, is due to old age or other reasons, such as congenital joint abnormalities, joint deformities, trauma and other causes of non-inflammatory articular cartilage degeneration and joint marginal osteophyte formation. It can produce clinical joint pain, limited mobility and joint deformities and other symptoms and severely affect the health of the aged people. In recent years, along with in-depth study the pathogenesis of OA, we gradually realize that the pathological process in OA is caused by inflammatory cytokines, such as IL-1βand TNF-a, which lead to articular cartilage damage and cartilage matrix degradation. By so far the treatment of OA performs unconveniently, suicide gene therapy for the treatment of osteoarthritis is a new approach. The pathological mechanism in rabbits can be used as animal models in the study of osteoarthritis. In this experiment we use New Zealand white rabbits to produce model of knee osteoarthritis and detect the content of inflammatory cytokines, such as IL-1β, TNF-a content changes, the time of peak levels of inflammatory cytokines in the process of cartilage degeneration and synovial proliferation. We also detect the expression of the pSNAV2. O-HSVl-tk-IRES-hIL-1Ra packaged by the adeno-associated virus in vivo. In this case, the system of HSVl-tk/GCV can inhibit synovial cell proliferation, and we can also use its'bystander effect'to kill the synovial cells far away. The expression of hIL-1Ra gene is to produce hIL-1Ra, which can inhibit or reduce the effect of IL-1, delaying the progress of OA.Our this research may lay a solid foundation for gene therapy of osteoarthritis in the future.Material and methods①We use New Zealand white rabbits which are 3-4 months old and Inject 3% pentobarbital anesthesia (lml/kg) vie ear vein, then clearly expose the right hind knee joint with its hair removal,In the sterile surgical operation, we have the anterior cruciate ligament and medial meniscus of knee removed. And inject penicillin 80 million units/day for three days.②Open the corresponding model of knee joint respectively after 2 weeks, 4 weeks,8 weeks,12 weeks. Take synovium, cartilage, synovial fluid and blood for further experiments.③Detect the synovial hyperplasia and cartilage degeneration with HE staining and also detect the content changes of IL-1βand TNF-a with ELISA in serum and synovial fluid after 2 weeks,4 weeks,8 weeks,12 weeks respectively.④Co-expression of recombinant vector pSNAV2. O-HSVl-tk-IRES-hIL-1Ra was kindly provided by Liu Qingchun; rAAV2/1-TK-IL-1αis the product of pSNAV2. O-HSVl-tk-IRES-hIL-1Ra packaged by adeno-associated virus. The solution of rAAV2/1-TK-IL-1αwas diluted to a concentration of 1.45X1011v. g./ml, then we inject 1ml this solution to the cavity of the model of the rabbit knee. Four weeks later, corresponding animals were killed quickly and we fetch the synovial tissue, cartilage, synovial fluid, blood as the experimental samples to do the experiment followed, such as HE staining, safranin 0 staining and ELISA.⑤Extract RNA from synovial specimens with Rapid Isolation of total RNA with column kit (RLT-centrifuge columnar), and add primer reverse transcription to generate cDNA. Then real-time quantitative PCR (RT-PCR) is used to detect the expression of PSNAV2. O-HSV1-tk-IRES-hIL-1Ra in the synovial tissue.Results①Two weeks after modeling:synovial is congestive, red and edema. Articular cartilage of the medial femoral condyle is dark, and there is no defect, failure or loss. Four weeks after modeling:Synovial is hyperemia and marked hyperplasia; cartilage degeneration is obvious, with lower elasticity and gray color. Eight weeks after modeling:synovial' hyperplasia is obvious, with congestion, edema and brittle texture; the surface of the cartilage is dull, rough and uneven, and also its elasticity decreases. Twelve weeks after modeling:synovial tissue is significant proliferation, with congestion and edema and significant cartilage de generat i on.②The results of histological sections:Two weeks after modeling, HE staining showed hyperemia and edema of synovial epithelium. The surface of cartilage become slightly uneven and cartilage cells began to appear the cell cluster phenomenon. Safranin 0 staining shows some staining of chondrocytes unevenly around the area. Four weeks after modeling:HE staining shows synovial epithelial hyperplasia and edema, with inflammatory cells infiltrating and mild proliferation of collagen fibers on the skin. Cartilage cells reduces, unevenly distributed, with cluster phenomenon. Eight weeks after modeling:HE staining shows marked hyperplasia of synovial epithelium, with inflammatory cell infiltration. The collagen fibers under the skin proliferate significantly. The number of chondrocyte which is necrosis Increases. With large cells cluster. Safranin 0 staining shows that cartilage matrix is stained light and the loss of stained area increases. Twelve weeks after modeling:. HE staining shows marked hyperplasia of synovial epithelium, with lots of inflammatory cells infiltrating. There are a lot of cartilage cells necrosis with significant cartilage fibrosis. And also there is angiogenesis and invasion of the tide line. Safranin 0 staining shows that the number of cartilage lacuna in the cartilage matrix increases significantly, and full-thickness cartilage staining disappears at some of the area.③The change of inflammatory cytokines, such as IL-1β, TNF-αcontent in rabbit knee osteoarthritis, is statistically significant. The concentration of IL-1βis the highest when it's two weeks after the operation of modeling, while the concentration decreases when it's four, eight or twelve weeks after the operation of modeling;but it is still higher than the control group. The concentration of TNF-αsignificantly increases until 4 weeks after the operation of modeling, while the concentration is lower when it's eight or twelve weeks after the operation;but it's still higher than the control group.④Inject the system of pSNAV2.0-HSV1-tk-IRES-hIL-1Ra which is packaged by adeno-associated virus-rAAV2/1-TK-IL-1αinto knee joint cavity to transfect articular synovial cells.Then detected by RT-PCR, we could find the specific bands, which indicates that cells transfected by rAAV2/1-TK-IL-1αcan express specific geneConclusion①It's a useful and convinent method to create a rabbit knee joint osteoarthritis model by cutting off the anterior cruciate ligament and medial meniscus. With the progression of osteoarthritis, synovial tissue proliferation and inflammatory degenerative changes in cartilage gradually increased.②The concentration of IL-1βis the highest when it's two weeks after the operation of modeling, while the concentration decreases when it's four, eight or twelve weeks after the operation of modeling;but it is still higher than the control group. The concentration of TNF-αsignificantly increases until four weeks after the operation of modeling, while the concentration is lower when it's eight or twelve weeks after the operation;but it's still higher than the control group.③The system of pSNAV2.0-HSV1-tk-IRES-hIL-1Ra packaged by adeno-associated virus-rAAV2/1-TK-IL-1αcan transfect articular synovial cells and express specific gene. |
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