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
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, 观看其在大鼠肝细胞系BRL中的表达, 比较有无受体介导的脂质体的转染效率。方式: 抽提外周血单个核细胞的总RNA, 通过RT-巢式PCR方式取得大鼠IL-10的全长编码序列, 定向克隆到真核表达载体, 并进行限制性内切酶酶切及测序鉴定。将重组质粒别离通过脂质体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方式检测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明, 重组质粒插入片段与大鼠IL-10的全长编码序列完全相符。发觉受体介导的脂质体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细胞取得高水平的IL-10表达。结论: 成功地构建重组质粒。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基因医治肝纤维化的有效转染载体。
【关键词】 白细胞介素-10 基因医治 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 . 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进展为肝硬化的一起病理进程。白细胞介素-10（IL-10）是一种具多效性的细胞因子, 在肝纤维化的进程中扮演重要的负反馈角色［1］。引入外源性IL-10也被证明对肝纤维化有拮抗作用［2］。可是由于重组IL-10半衰期很短, 难以在体内维持一个相对稳固的浓度; 静脉用药, 其作用缺乏肝脏靶向性。而探讨通过适合途径进行IL-10的基因医治, 那么有可能解决上述问题, 具有必然的应用前景。本实验中构建大鼠IL-10基因的真核表达载体, 并通过受体介导的脂质体和非受体介导的脂质体转染大鼠肝细胞系BRL, 观看IL-10的表达情形, 比较2种有无受体介导的脂质体的转染效率, 为下一步IL-10基因医治肝纤维化的动物实验奠定了基础。
1 材料和方式
材料 真核表达载体由福建省消化研究室保留; 逆转录酶购自Fermentas公司; 高保真Taq酶、 限制性内切酶Hind Ⅲ、 BamHⅠ及T4连接酶均购自Promega公司; 质粒抽提试剂盒购自Qiagen公司; 胶回收试剂盒购自上海中科开瑞生物芯片科技; PEIjet-gal转染载体购自polyplus公司; TransfastTM脂质体购自Promega公司, RNA抽提试剂盒购自Gentra公司; 大鼠IL-10 ELISA试剂盒购自
大鼠IL10基因真核表达质粒的构建及其在BRL细胞中的表达
