真核表达质粒pCMV-脂联素的构建及其转染对高糖环境下肾系膜细胞增殖影响的研究

发布时间：2018-06-02 12:44

本文选题：真核表达质粒 + pCMV-脂联素　；参考：《中国糖尿病杂志》2017年07期

【摘要】：目的构建pCMV-脂联素(APN)真核表达质粒,观察转染小鼠肾系膜细胞后APN的表达以及对高糖环境下肾系膜细胞增殖的影响。方法通过RT-PCR扩增小鼠肾系膜细胞中APN基因片段,经酶切、纯化后与pcDNA3.1质粒连接构建pCMV-APN重组质粒。pCMV-APN重组质粒经酶切鉴定及DNA测序验证后,由脂质体转染将pCMV-APN真核表达质粒转导入肾系膜细胞中,Western blot检测转染后细胞中APN蛋白表达,MTT检测转染后细胞在高糖环境下增殖活性的变化。结果真核表达质粒pCMV-APN经酶切、测序鉴定后提示构建成功。转染后肾系膜细胞APN蛋白表达明显增高,且在高糖环境下的增殖活性较空载体转染组明显下降[(0.87±0.06)vs(0.60±0.01),P0.05]。结论成功构建pCMV-APN真核表达质粒,能稳定表达于小鼠肾系膜细胞及抑制高糖的诱导增殖作用,为进一步研究APN的生物学功能奠定理论基础。
[Abstract]:Objective to construct the eukaryotic expression plasmid of pCMV- adiponectin (APN) and observe the expression of APN and its effect on the proliferation of Mesangial cells in high glucose environment. Methods the APN gene fragment of mouse Mesangial cells was amplified by RT-PCR. The recombinant plasmid pCMV-APN was ligated with pcDNA3.1 plasmid and digested by enzyme. The recombinant plasmid pCMV-APN was identified by restriction endonuclease digestion and confirmed by DNA sequencing. The eukaryotic expression plasmid of pCMV-APN was transfected into Mesangial cells by liposome transfection. Western blot was used to detect the expression of APN protein in the transfected cells. Results the eukaryotic expression plasmid pCMV-APN was digested by enzyme and identified by sequencing. The expression of APN protein in renal Mesangial cells was significantly increased after transfection, and the proliferative activity in high glucose environment was significantly lower than that in empty vector transfection group [0.87 卤0.06)vs(0.60 卤0.01 p0.05]. Conclusion the successful construction of pCMV-APN eukaryotic expression plasmid can stably express in mouse Mesangial cells and inhibit the proliferation induced by high glucose, which lays a theoretical foundation for the further study of the biological function of APN.
【作者单位】：福建省老年医院内分泌科;福建省立医院内分泌科;福建医科大学附属协和医院内分泌科;
【基金】：福建省医学创新课题(2015-CX-13)
【分类号】：R587.2;R692.9

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