Experimental Study Of Intra-articular Injection Of Retroviral Vector Mediated By The Recombinant Human Il-1ra And Tgf-¦Â1 Gene Therapy Rabbit Knee Osteoarthritis

Osteoarthritis (OA) is a chronic degenerative joint disease, which is common in the elderly group especially in women. It seriously affected quality of life of patients and consumed a large amount of family and social resource. Once articular cartilage is injured, it has a very limited capacity for self-repair. Although current surgical therapeutic procedures to cartilage repair are clinically useful, they cannot reconstruct a normal articular surface. The idea of modern therapeutic techniques is to achieve a more hyalinelike cartilage repair tissue by transplanting tissues or cells. Tissue transplantation procedures such as periosteum, perichondrium, or osteochondral grafts have shown positive short term effect in a number of patients, but the long term clinical effect is still uncertain, and, tissue availability may be a major limitation for transplanting, especially in large cartilage defects. For this problem, tissue engineering may be a good choice, which is being aggressively investigated in an effort to cultivate cartilage by tissue engineering techniques in vitro to produce grafts to facilitate regeneration of articular cartilage in vivo. The key to this process is gene transfer technology, the application of which is pioneered as a means to treat arthritis. In all joints receiving gene therapy, intra-articular gene transfer and expression were observed. These cases suggest that gene transfer may have optimistic outlook in treating articular disorders for which current treatment modalities are unsatisfactory. Ideally, Gene should expressed continuously at damage region. Gene transfer can be achieved by either direct vector administration to cells located at or surrounding the damage region, or transplantation of genetically modified chondrogenic cells into the damage region. In vitro the procedures are generally more invasive, expensive and technically tedious. The transfection efficiency and safety testing are important prior to transplantation. In vivo the procedures are simpler, cheaper, and less invasive, but in this process viruses are introduced directly into the body, the safety of which is still controversial. Direct intra-articular injection of vector or modified cells results in synthesis and release of therapeutic proteins into the joint space, which then bathe all tissues, including injured cartilage. In animal models, encouraging results have been reported for adenovirally delivered IGF-1or IL-1Ra for OA and localized cartilage injury. In some cases that Adenoviral mediated delivery of TGF-β1 or BMP-2 to the synovial lining has been applied, joint fibrosis, extreme swelling, osteophytes and cartilage degeneration are found. The results have shown promising outlook in maintaining and promoting the chondrogenic phenotype in vivo, including TGF-β1, BMP-2,-4,-7, IGF-1,SOX9 among others. In clinical application, the test in cell and animal experiments is needed to confirm which genes or gene combinations will produce expected effect. In this study, we use retrovirus vector, which recombine human IL-1Ra (IL-1 catabolic factor receptor antagonist) and TGF-β1(anabolic important factor) gene to transfect normal chondrocytes and OA chondrocytes. And chondrocytes proliferation were observed. Intra-articular injection which directly injected genes into the animal model of knee osteoarthritis, expects to promote the regeneration of the chondrocytes and extracellular matrix synthesis and refrain the degradation of chondrocytes and extracellular matrix.PartⅠEffects of co-transfection of recombined human IL1-Ra and TGF-β1 on rabbit chondrocytes proliferation in vitro.Objective:To investigate the effects of co-transfection of IL-1Ra and TGF-β1 on rabbit chondrocytes and OA chondrocytes mediated by retrovirus vector.Methods:Cultured normal and OA chondrocytes are divided into the Control group, empty vector transfected group (PLNCX2), IL-1Ra single-gene transfer group, TGF-(31 single-gene transfer group and IL-1Ra and TGF-β1 combined gene transfer group. Transient gene expression was detected respectively through Enzyme-linked immunosorbent assay (ELISA).Results:In control group and PLNCX2 empty vector transfected group the difference was not statistically significant (P>0.05); There is a significantly gene expression in gene transfected group(P<0.05); In combined-gene transfected group levels of the IL-1Ra and TGF-β1 were significantly increased compared with single-gene transfected group (P＜0.01); In IL-1Ra single gene transfected group the expression of gene between normal chondrocytes and OA chondrocytes was not statistically significant (P>0.05), while in the combined gene transfected group there was a significant difference (P<0.05); In TGF-β1 single gene transfected and combined gene transfected group there was no significant difference between the normal chondrocytes and OA chondrocytes (P＞0.05).Conclusion:The expression of IL1-Ra and TGF-β1 genes have been detected after being transfected into chondrocytes and the biologic activity of chondrocytes transfected with combined gene is beyond single gene. The expression of OA chondrocytes is weaker than normal chondrocytes in IL-1Ra, TGF-β1 single-gene and combined-gene transfer group.PartⅡThe effects of gene therapy of osteoarthritis through intra-articular injection of retroviral vector-mediated recombinant human IL-1Ra and TGF-β1Objective:To observe the effects of intra-articular injection of retrovirus-mediated interleukin-1 receptor antagonist (IL-1Ra) and transforming growth factor-β1 (TGF-β1) gene expression on the treatment of osteoarthritis.Methods:30 NZW rabbits were distributed to 5 groups randomly,Ⅰgroup was the control group (no ACLT processing),others establishing model of osteoarthritis through anterior cruciate ligament transaction (ACLT):Ⅱgroup no gene injected; InⅢgroup Virus suspension which integrated IL-1Ra was injected into articular cavity. InⅣgroup TGF-β1 injected,Ⅴgroup IL-1Ra and TGF-β1 combined gene injected. After the injection of foreign genes 4 and 8 weeks, the synovial fluid is abstracted for ELISA analysis, the joint specimens for Mankin's score, Alcian-blue-periodic acid Schiffreagent (AB-PAS)staining, TGF-β1, IL-1Ra and typeⅡcollagen in situ hybridization and immunohistochemistry.Results:Four and eight weeks after intra-articular injection, the control group in general score and Mankin's score is significantly lower than the operated group (P<0.01); the general score and Mankin's score ofⅡgroup was higher than gene transfer group(P<0.05); Compared with 4 weeks,8 weeks after gene therapy the general score and Mankin's score of all groups increase, but there is no significant difference. Synovial fluid ELISA analysis shows that there is no significant difference beweenⅠgroup andⅡgroup (P>0.05); and the difference is Statistically significant between TGF-β1, IL-1Ra single-gene transfer group and combined-gene transfer group (P＜0.05); 4 weeks after injected, between the single-gene transfer group and combined-gene transfer group, the difference was statistically significant (P＜0.05); 8 weeks, no significant difference statistically (P>0.05); Compared with 4 weeks, the genetic content of 8 weeks after injected have reducted, the difference was significant (P＜0.05). The cytokine staining ofⅢ-Ⅴgroup is more deeper thanⅠgroup andⅡgroup, the differences of gray value were significantly different (P<0.01),and beween the single-gene transfer group and double-gene transfer group the differences in gray values were significantly different (P<0.01); 8 weeks after gene therapy, the positive staining cells is less than 4 weeks and there are significantly different (P<0.05).8 weeks after injecting the expression of gene weakened.Conclusion:Retroviral vector mediated genes injected to rabbit knee joint cavity have obtain stable expression and the expression of TGF-β1 and IL-1Ra can effectively prevent the further development of OA; Combined gene transfer showed better efficacy compared with single gene transfer, and the timeliness of the expression have been recognized.