Effects Of Co-transfection Of Recombined Human IL1-Ra And TGF-β1 On Rabbit Chondrocytes Proliferation Ex Vitro.

Osteoarthritis is an ordinary disease which impact aged people commonly. Morphologic changes observed in osteoarthritis include cartilage erosion and a variable degree of synovial inflammation.Current research attributes these changes to a complex network of biochemical factors which lead to a breakdown of the cartilage macromolecules.Despite an extensive armamentarium and numerous surgical options, osteoarthritis remains incurable because of the poor repair ability of articular cartilage, and an improved approach in the treatment is imperative.Though the favorable biologic properties of manygrowth factors,their clinical application is hindered by their short half-life and delivery problems.Inparticular,it is difficult to maintain therapeutic levels of growth factors locally within the lesion for the length of time that is likely to be needed to achieve repair.To improve cartilage healing need slow-release growth factors.Recent advances in molecular biology indicate that gene therapy may provide a novel potential approach for the procedure of slow-release.The vectors encoding specific genes can deliver them to suitable sites under conditions in which gene expression remains at therapeutic levels for the appropriate periods of time. As a prelude to studies,it is necessary to determine which gene,or combinations of genes,gives the best result.We used recombined human IL1-Ra and TGF-β1 genen the present experiment.PartⅠEffects of eo-transfeetion of recombincd human IL1-Ra and TGF-β1 on rabbit chondrocytes proliferation ex vitro.Objective To investigate the effects of co-transfection of IL-1Ra and TGF-β1 on rabbit chondrocytes mediated by retrovirus vector.Methods Monolayer cultures of rabbit articular chondrocytes were infected with recombinant plasmid PLNCX2 carrying genes encoding the following growth factors respectively:IL 1-Ra and TGF-β1.The synthesis of IL- 1Ra,TGF-β1 and typeⅡcollagen was measured by enzyme-linked immune- osorbent assay(ELISA),immuneohistochemistry, flow cytometer.Results The positive expression was detected in the cell culture supernatant. The content of IL-1Ra,TGF-β1 and typeⅡcollagen in double gene transfection group was highly elevated.the expression of TGF-β1 and typeⅡcollagen in IL-1Ra gene transfection group was less than double gene transfection group(P＜0.05)and IL-1Ra in TGF-β1 gene transfection group was less(P＜0.05).The chondrocytes at stage S in gene transfection group was obviously more than that in blank control group.Gonclusion IL1-Ra and TGF-β1 genes could be expressed effectively after being transfected into chondrocytes,the biologic activity of chondrocytes transfected with double gene beyond single gene.The results encourage the further development of gene therapy for osteoarthritis.PartⅡEffects of co-transfection of recombined human IL1-Ra and TGF-β1 on osteoarthritis of rabbit knee articular cartilagon ex vivo.Objective To decide whether recombined human IL1-Ra and TGF-β1 gene have positive influences on ACLT-induced.osteoarthritis-like changes in NZW ra bbit articular.Methods 30NZW rabbits were distributed to 5 groups randomly after been caused osteoarthritis by anterior cruciate ligament transection(ACLT),and ano ther six rabbits belong to normal control group(group 1).Recombined TGF-β1 gene and/or IL1-Ragene(group 6-8),chondrocytes which been transfected with TG F-β1 gene and/or IL1-Ra gene(group 3-5)were injected into knee articulars of th ese NZW rabbits.Experimental control group(group 2)was only suffered ACLT but been injected nothing.After 4,8weeks,rabbits were sacrificed and evaluated by morphological grades,histological examination,and the examination of in situ hybridization,immunohistochemistry.Results The data of morphological grades showed that the normal contro 1 had a very significant difference with experimental control group(P＜0.01),the group of been injected chondrocytes carring TGF-β1 gene and the group of bee n injected double recombined genes had a significant difference with experiment al control group(P＜0.05).The in situ hybridization and immunohistochemistry e xamination showed the same results with above-mentioned,and the group of been injected chondrocytes carring TGF-β1 gene had a significant difference with the group of been injected recombined IL1-Ragene(P＜0.05).After 8 weeks,the above examination data showed that all groups lower than the data of 4 weeks except t he normal control groupand experimental control group(P＜0.05),control group aft er gene therapy,but it was more turbulent after 8 weeks than that after 4 weeks.Conclusion It is effectual on osteoarthritis to inject recombined TGF-R1, IL1-Ra genes and inject chondrocytes carringrecombined TGF-β1,IL1-Ra genes into NZW rabbits knee joints.The therapic effect of groups 3-5 was better than that of groups 6-8,and it was obvious that the therapic effect of double genes was b etter than that of single gene.The fact that gene expression was decreased gra-d ually after 4 weeks makes out that gene therapy is limited by time.These resultss uggest that therapeutic TGF-β1 and IL1-Ra gene transfer may be applicable for th e treatment of OA.