大鼠IL-10基因真核表达质粒的构建及其在BRL细胞中的表达_临床医学论文.docVIP

大鼠IL-10基因真核表达质粒的构建及其在BRL细胞中的表达_临床医学论文 大鼠IL-10基因真核表达质粒的构建及其在BRL细胞中的表达_临床医学论文 作者：陈运新, 黄月红, 陈治新, 郑伟达, 王小众 【摘要】 　　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-10 mRNA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。 【关键词】 白细胞介素-10 基因治疗 去唾液酸糖蛋白受体 肝细胞 　　［Abstract］ AIM: To construct eukaryotic expression vector of rat IL-10 gene and observe its expression in hepatocyte cell line BRL. METHODS: Total RNA was extracted from rat peripheral blood mononuclear cells. The full length coding region of IL-10 was amplified by RT- nested PCR and cloned into eukaryotic expression vector pcDNA3.0. The recombinant plasmid was transfected into BRL cells with either liposome TransfastTM or asialoglycoprotein receptor mediated liposome PEIjet-gal respectively. The expression of IL-10 mRNA was detected with PCR and that of IL-10 secreted from BRL cells transfected by liposome PEIjet-gal was detected with ELISA. RESULTS: The recombinant plasmid was identified and confirmed with digestion of restriction endonuclease and DNA sequencing. Receptor mediated liposome PEIjet-gal exhibited significantly higher transfection efficiency than liposome TransfastTM and higher level secretory IL-10 expressed in BRL cells. CONCLUSION: The eukaryotic expression vector of IL-10 gene was successfully constructed. Asialoglycoprotein receptor-mediated liposome had high transfection efficiency on hepatocytes, suggesting that it could be a potential hepatocyte-targeting delivery system for IL-10 gene therepy. 　　［Keywords］interleukin-10; gene therapy; asialoglycoprotein receptor; hepatocyte 　　肝纤维化是多种慢性肝病发展为肝硬化的共同病理过程。白细胞介素-10（IL-10）是一种具多效性的细胞因子, 在肝