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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),P<0.05]。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。
Objective To construct eukaryotic expression plasmid pCMV-adiponectin (APN) and observe the expression of APN and the effect on the proliferation of mesangial cells under the high glucose condition in transfected mouse mesangial cells. Methods The APN gene fragment of mouse mesangial cells was amplified by RT-PCR. After digestion, the recombinant plasmid was ligated with pcDNA3.1 plasmid to construct pCMV-APN recombinant plasmid. The pCMV-APN recombinant plasmid was verified by restriction enzyme digestion and DNA sequencing. The pCMV-APN eukaryotic expression plasmid was transfected into mesangial cells by lipofectamine. The expression of APN protein was detected by Western blot, MTT The changes of proliferation activity of the transfected cells under high glucose condition were detected. Results The eukaryotic expression plasmid pCMV-APN was confirmed by restriction analysis and sequencing. The expression of APN protein in renal mesangial cells was significantly increased after transfection compared with that in empty vector transfected cells ([0.87 ± 0.06] vs (0.60 ± 0.01), P <0.05]. Conclusion The eukaryotic expression plasmid pCMV-APN was successfully constructed and could stably express in mesangial cells of mouse and inhibit the proliferation induced by high glucose, which laid a theoretical foundation for further study on the biological function of APN.