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目的:构建大鼠IL-10基因的真核表达载体,观察其在大鼠肝细胞系BRL中的表达,比较有无受体介导的脂质体的转染效率。方法:抽提外周血单个核细胞的总RNA,通过RT-巢式PCR方法获得大鼠IL-10的全长编码序列,定向克隆到真核表达载体pcDNA3·0,并进行限制性内切酶酶切及测序鉴定。将重组质粒分别通过脂质体TransfastTM与去唾液酸糖蛋白受体介导的脂质体PEIjet-gal转入大鼠肝细胞系BRL,通过RT-PCR方法检测IL-10mRNA的表达,比较二者的转染效率,用ELISA法检测后者分泌型IL-10的表达。结果:经酶切及测序鉴定证实,重组质粒插入片段与大鼠IL-10的全长编码序列完全相符。发现受体介导的脂质体PEIjet-gal转染效率明显高于非受体介导的脂质体TransfastTM。通过受体介导的脂质体转染,BRL细胞获得高水平的IL-10表达。结论:成功地构建pcDNA3·0-IL-10重组质粒。受体介导的脂质体对肝细胞有较高的转染活性,可能成为IL-10基因治疗肝纤维化的有效转染载体。
OBJECTIVE: To construct the eukaryotic expression vector of rat IL-10 gene and observe its expression in rat hepatocyte cell line BRL, comparing with or without receptor-mediated transfection efficiency of liposome. Methods: The total RNA of peripheral blood mononuclear cells was extracted. The full-length coding sequence of rat IL-10 was obtained by RT-nested PCR and cloned into the eukaryotic expression vector pcDNA3.0. The restriction endonuclease Digestion and sequencing identification. The recombinant plasmids were transfected into the rat hepatocyte cell line BRL by liposome PEIjet-gal mediated by liposome TransfastTM and asialoglycoprotein receptor respectively, and the expression of IL-10 mRNA was detected by RT-PCR. The transfection efficiency of the latter was tested by ELISA for the expression of secreted IL-10. Results: After digestion and sequencing, it was confirmed that the inserted fragment of recombinant plasmid was completely consistent with the full-length coding sequence of rat IL-10. The receptor-mediated liposome PEIjet-gal transfection efficiency was found to be significantly higher than that of the non-receptor-mediated liposome TransfastTM. BRL cells obtained high levels of IL-10 expression by receptor-mediated lipofection. Conclusion: The pcDNA3.0-IL-10 recombinant plasmid was successfully constructed. Receptor-mediated liposomes have high transfection activity on hepatocytes, which may be an effective transfection vector for the treatment of hepatic fibrosis with IL-10 gene.