vIL-10转基因治疗兔膝关节炎模型的实验研究

Study on the animal model of vIL-10 transgenic therapy for rabbit arthritis

目的以逆转录病毒重组体rRV-vIL-10为治疗基因,通过体内外基因转移的动物实验研究,建立局部兔膝关节炎间接体内基因治疗方法。方法①利用可以稳定表达hIL-1β的兔滑膜原代细胞(MFG-hIL-1β-neo-HIG-82)诱导出兔膝关节炎模型。②在体内实验中,将在体外培养的已转染rRV-vIL-10的兔关节滑膜细胞经G418筛选出阳性细胞后回输注入兔膝关节腔内,进行间接体内基因治疗。③通过RT-PCR方法和免疫组化法证实治疗基因转移至体内的滑膜组织并有效表达目的蛋白。④ELISA方法测定关节炎的相关细胞因子在基因治疗前后的水平变化。结果①通过RT-PCR及免疫组化的方法证实逆转录病毒重组体rRV-vIL-10能有效转染兔膝关节的滑膜组织。②应用rRV-vIL-10重组体进行局部间接体内基因治疗可以明显改善hIL-1β诱导的兔膝关节炎模型的关节炎症,下调炎症相关的细胞因子hIL-1β的水平。结论逆转录病毒重组体rRV-vIL-10可以成功地将vIL-10基因导入兔滑膜成纤维样细胞和滑膜组织,明显降低hIL-1β诱导的兔膝关节炎的炎症水平。

Objective：To establish a local ex-vivo gene transfer method to treat RA through animal experiments in vivo and in vitro using retrovirus（rV） as a vector which carrying rRV-vIL-10 target gene. Methods ： ①The rabbit RA models were induced by the rabbit synovial fibroblast cell line which could continuously expreesed hIL-1β. ②In vivo, the rabbit synovial fibroblast cell line was transduced with rRV-vlL-10, then adding G418 to pick out the rRV-vIL-10 positive clon and infecting the rabbit synovium through intra-articular injection. ③RT-PCR, IHC methods were performed to prove the success of gene transfer to the rabbit synovium and expressed the target protein. ④The relative cytokins changes were detected by ELISA before and after gene therapy and evaluated treatment efficacy of rRV-vIL-10. Results ：①rRV-vIL-10 was a effective vector which could transfect to the rabbit synovium in vivo through RT-PCR and IHC methods. ②Intra-articular local gene therapy could effectly reduce the synovium inflaroJnation level of rabbit joints and expressed mRNA and vIL-10 protein. The level of cytokin such as IL-1β was decline. Conclusion ： Retrovirus-mediated transgene of vIL-10 is successfully transfected to the rabbit synovium ex-vivo and can reduce arthritis inflammation levels of the IL-1β induced ar thritis.