| Objective To investigate the effects and mechanisms of HSV-1based vector mediated interlukin-1receptor antagonist (IL-IRa) gene transfer for injections in treating rabbit model of osteoarthritis.Methods HSV-1vectors containing IL-1Ra genes were constructed and then injected into the joint space of the rabbit osteoarthritis knee. Three days after injection of the pHSV-IL-1Ra-LacZ and pHSV-LacZ. At week2, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and IL-1expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stmned with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated. To investigation the regulation mechanism of IL-IRa in OA cells. OA cells were transfected with pHSV-IL-1Ra-LacZ fusion protein recombinant plasmid in vitro. Then, the OA cells were incubation different concentrations of TNF-alpha (Ong/mL,5ng/mL,10ng/mL,15ng/mL and25ng/mL). The expression level of IL-1Ra, TNF-α and IL-1were detected by real-time quantitative PCR and Western blot.To explore the molecular mechanism of IL-1Ra expression is affect on regulation signaling pathway. The P38, ERK, JNK and PI3K signaling pathway were inhabit on with corresponding signaling pathway-specific inhibitor blocked (U0126, SB203580, of SP600125, LY294002respectively).Results Intraarticular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. The results of digestion and sequencing of Construction of recombinant plasmid pHSV-IL-1Ra-LacZ showed that plasmid with over-expression IL-1Ra by the use of recombinant DNA technology requirements to comply with following experiments. In transfected cells, At the same time, using quantitative PCR and Western blot, we verified IL-1Ra mRNA and protein expression in exogenous recombinant plasmid pHSV-IL-1Ra-LacZ cells were higher than the control group (P<0.05). We next to used flow cytometry to test the effect of over expression of IL-1Ra on cells. The results showed that overexpression of IL-1Ra promotes cell apoptosis (P<0.05). Using Real time quantitative PCR and Western blot test methods. Furthermore, the expression of IL-1Ra were decreased when simultaneously induced by TNF-a (P<0.05). Thus, TNF-a induced can be control the expression of IL-1Ra. Results showed that the expression of IL-1, IL-6, IL-8inflammatory factors were decreased significantly when using SB203580inhibition. The reauluts indicated IL-1Ra has regulated the expression of inflammatory factors mainly through signal transduction of P38pathway. when using PD 98059inhibited ERK pathway, the inflammatory factor expression was also apparent,but not as good as P38pathway. The PBEF mainly regulated the expression of inflammatory cytokines through P38pathway signaling, and was possibly just a small part invoved in JNK signal transduction pathwayConclusion These results suggesting IL-1Ra may play important role in osteoarthrotis.Targeting IL-1Ra up regulated the expression of IL-1Ra, the expression of IL-1, IL-6and IL-8.The IL-1Ra regulated the expression of osteoarthritis inflammatory... |
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