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  

Construction and tansfection of pCMV-APN eukaryotic expression plasmid and its effect on high glucoseinduced mesangial cells proliferation

作　　者：冯敏 温俊平[2] 吕琦 黄国良[3] 

机构地区：[1]福建省老年医院内分泌科,350003 [2]福建省立医院内分泌科 [3]福建医科大学附属协和医院内分泌科

出　　处：《中国糖尿病杂志》2017年第7期649-654,共6页Chinese Journal of Diabetes

基　　金：福建省医学创新课题(2015-CX-13)

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),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 an eukaryotic expression plasmid pCMV-adiponectin（APN）and to detect its expression in mouse mesangial cells and the influence of its expression on high glucose-induced cell proliferation. Methods APN gene was amplified by RT-PCR with cDNA in mouse mesangial cells,as the template and the fragment was combined with plasmid pcDNA3.1.The recombinant eukaryotic expression plasmid pCMV-APN was transfected into mesangial cells by using Lipofectamine 2000.The expression level of APN after transfection was detected by Western blot.The influence of its expression on high glucose-induced cell proliferation was analyzed by MTT. Results After identification by restriction enzyme digestion and sequencing,the eukaryotic expression plasmid pCMV-APN was successfully constructed.The expression level of APN in mouse mesangial cells transfected by pCMV-APN was significantly higher compared with control group and pcDNA3.1 transfected group.Meanwhile,MTT testing showed that mesangial cells proliferation was inhibited after being transfected with the recombinant plasmid compared with control group and pcDNA3.1 transfected group[（0.87±0.06） vs （0.60±0.01）,P〈0.05]. Conclusion The eukaryotic expression plasmid pCMV-APN is successfully constructed,stably expressed in mouse mesangial cells and inhibited cell proliferation,which lays the foundation for the further study of the biological functions of APN and protective effect of diabetic nephropathy.

关 键 词：真核表达质粒 pCMV-脂联素 肾系膜细胞 转染 细胞增殖 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]