A Combinatory Strategy For Gene Therapy Against OA: IL-1Ra/HSV-tk Transferred To Synoviocytes In Vitro Reduced The Release Of Proinflammatory Cytokine As Well As Inhibited Proliferation Of Synoviocytes

Objective:Osteoarthritis (OA) is a prevalent health problem that particularly affects daily life of elder people and causes tremendous burden mentally and economically to the individuals and the society. The epidemiology shows no apparent ethnicity or regional features, and the pathogenesis involves changes on morphology, biochemistry and metabolism. In OA, excessive release of proinflammatory cytokines attributes greatly to the pathological changes in local joint. Among these cytokines, IL-1 and TNF undoubtedly play the pivotal role. A series of treatments against OA were developed according to the biological functions of the cytokines. As this studying field progressed, Synovial tissue has been focused on more and more in pathological changes of knee OA for its release of proinflammatory cytokines and the abnormal hyperplasia, which cause degradation, swelling in local joint and triggered series of clinical symptoms. In this study, we adopted gene therapy and used rAAV carrying the anti-inflammatory cytokine gene combined with suicide gene to transfect the synoviocytes in vitro, trying to reduce the release of proinflammatory cytokines and inhibit the proliferation of synoviocyte for the treatment against OA.Methods:In this study, we applied the strategy of anti-cytokines combined with inducing death of target cells according to OA pathological features. We used the viral vector rAAV, carrying IL-1Ra and suicide gene HSV-tk and made comparisons evaluations of efficacies among single gene transferred groups that were separately transferred IL-1Ra gene as Group A, HSV-tk gene as Group B as well as combined-gene transferred Group C and the control group D, being transfected by the rAAV carrying no target gene. All the experiments in this study followed the same rules on group unless otherwise stated. First of all, we transduced the report gene-EGFP carried by rAAV to synoviocytes in vitro for observing the transduction capacity of rAAV and optimizing the transduction conditions. In terms of the cytotoxicity of GCV, we set two independent experiments including HSV-tk(+) test and HSV-tk(-) test. There were five concentrations of GCV,0.1,1,10,100,1000 (unit:μg/ml) and the length of time was from 24h to 96h with the interval of 24h. The bystander effect was also examined by MTT assay for the following experiments. The expressions of target genes including IL-1Ra, IL-1β, TNF-a, HSV-tk were measured by real-time PCR whereas the cytokines including IL-1Ra,IL-1β,TNF-a were measured by ELISA. Last of all, the apoptosis of synoviocytes induced by suicide gene HSV-tk was assayed by Flow Cytometry, using AnnexinⅤ-FITC-PI.Results:According to the results of cytotoxicity of GCV with HSV-tk(+) and the bystander effect tested by MTT assay, we found that after 72 hours of being transferred HSV-tk gene, GCV of 100μg/ml with ratio of 50% of co-cultured HSV-tk (+) optimized the desired results, the data were shown as mean±S.D, n=3 and P<0.05. According to the measurement of real-time PCR and the analysis of relative quantification-comparative CT method, The expression of IL-1Ra in Group A and C were respectively 66.49±10.85 and 117.63±21.88, which were significantly increased compared with non-transfected groups. Correspondingly, IL-1βin Group A, B and C all significantly decreased to 0.028±0.017,0.102±0.034 and 0.017±0.005. It was also detectable of significant expressions HSV-tk:252.663±53.673 in Group B and 513.867±120.821 in Group C. However, the obvious change did not occurred to TNF-a among three experimental groups, which were 3.563±2.511,5.523±2.285 and 1.017±0.162. Such trends were also notable at protein level for IL-1Ra, IL-1βand TNF-αby the ELISA assays. IL-1Ra was increased to 56.723±6.563pg/ml in Group A, 30.790±8.431pg/ml in Group B and 76.277±10.380pg/ml in Group C while it was 11.467±3.657pg/ml in Group D. IL-1βwas correspondingly decreased to 14.343±4.232pg/ml in Group A,21.300±5.112pg/ml in Group B and 6.534±2.353pg/ml in Group C, compared with 52.28±9.6367pg/ml in Group D. TNF-a also lacked changes after whatever being transferred anti-inflammatory cytokine and (or) suicide gene. TNF-αin three experimental groups (A, B, C) were respectively 9.733±5.804pg/ml, 14.247±2.255pg/ml and 6.527±3.785pg/ml and was 4.637±2.799pg/ml in Group D. The results of real-time PCR and ELISA were shown as mean±SEM, n=3 and P<0.05. Flow Cytometry was performed for the test for apoptosis of target cells induced by HSV-tk and found that the apoptotic rate in Group B was 65.40%, and was 69.40% in Group C, compared with 7.88% in Group A and 6.89% in Group D.Conclusions:The target genes were successfully transferred to the synoviocytes in vitro carried by rAAV. Although further evidences of anti-TNF-a are needed both in ex-vivo and in-vivo studies, the strategy has been proved superior by in-vitro study for the treatment against OA with the respect of anti-inflammatory cytokines and inhibiting proliferative synoviocytes.