弓莉;王元占;朱玉峰;吴湘慧;曾俊岭;刘谋荣;

• 1:南方医科大学南方医院实验动物研究中心
• 2:南方医科大学南方医院肾内科

摘要(Abstract)：

目的构建重组Toll样受体7(Toll-ike receptor 7,TLR7)真核表达质粒,并分析其对髓样树突状细胞(dendritic cell,DC)免疫功能的影响。方法用C57BL/6小鼠脾细胞制备原代DC,提取DC总RNA,以其逆转录合成的c DNA为模板扩增TLR7基因,克隆至载体pc DNA3.1(+)中,构建真核表达质粒pc DNA3.1-TLR7。经脂质体Lipofectamine2000将真核表达质粒转染至小鼠DC,经G418筛选阳性克隆。分别采用Real-time PCR及Western blot法检测转染细胞中TLR7基因m RNA转录及蛋白的表达水平;同时采用流式细胞术及细胞因子试剂盒检测转染细胞表面共刺激分子CD80、CD86和MHCⅡ的表达及分泌白细胞介素-6(interleukins-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的水平。结果经PCR及双酶切鉴定证明重组真核表达质粒pc DNA3.1-TLR7构建正确。转染12、24、48及72 h的DC中均可见TLR7基因的转录及蛋白的表达,且48 h时的转录及表达水平最高。转染48 h后,DC表面共刺激因子CD80、CD86和MHCⅡ的表达水平及其分泌IL-6和TNF-α的水平均显著提高(P<0.05)。结论成功建立了重组TLR7真核表达质粒,且其可明显提高DC的免疫功能。

关键词(KeyWords)： Toll样受体7;真核细胞;基因表达;树突状细胞;免疫功能

Abstract:

Keywords:

基金项目(Foundation): 广东省科技计划项目(2014A030304024);广东省科技计划项目(2013B060300012);; 南方医院院长基金项目(201213006)

作者(Author): 弓莉;王元占;朱玉峰;吴湘慧;曾俊岭;刘谋荣;

Email:

DOI: 10.13200/j.cnki.cjb.001319

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