人内皮脂酶584C→T变异型真核稳定表达载体的构建及其对人脐静脉内皮细胞生长的影响

Construction of the Stable Eukaryotic Expression Vector of Human Endothelial Lipase 584C→T Variation and Its Effect on the Growth of HUVECs

目的:构建人内皮脂酶(endothelial lipase,EL)野生型和584C→T变异型真核稳定表达载体及相应的转染细胞模型,为研究584C→T变异对内皮脂酶功能的影响奠定基础。方法:从pMIG/hEL及pMIG/hEL584C→T中双酶切分离出目标片段,与pCMV-N-his载体连接,凝胶电泳、双酶切电泳及测序鉴定构建的野生型和584C→T变异型内皮脂酶载体,而后以Lipofectamine LTX&PLUS为转染试剂,将其转入人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),G418筛选出稳定表达细胞,荧光定量PCR法检测转染细胞的EL表达,同时观察细胞形态,并用CCK-8法检测转染对细胞生长及细胞倍增时间的影响。结果:凝胶电泳及双酶切电泳结果显示条带位置与预期符合,测序结果表明其序列与GenBank上序列一致。荧光定量PCR法及mRNA电泳均显示:野生型和584C→T变异型转染细胞的EL表达显著高于空载体转染细胞。进一步研究584C→T变异对HUVEC细胞生长的影响,结果表明空载体、野生型EL和584C→T变异型EL转染HUVEC细胞与正常细胞的形态基本一致,且生长曲线和细胞倍增时间也无显著差异。结论:本实验成功构建了人内皮脂酶野生型和584C→T变异型真核稳定表达载体及相应的转染内皮细胞模型。EL584C→T变异对HUVEC细胞生长无明显影响。

Objective： To construct the stable eukaryotic expression vectors and the transfected cell models of wild type and 584C→T variation of human endothelial lipase （EL）. Methods： The wild and mutational EL fragment were isolated by double digestion from pMIG/hEL and pMIG/hEL584C→T were ligated with the digested pCMV-N-his vector. Restriction and sequencing were used to identify the constructional vectors of wild type and 584C→ T variation. Human umbilical vein endothelial cells （HUVECs） were transfected by the wild EL and 584C→T mutational EL with Lipofectamine LTX ＆ PLUS transfection reagent were screened by G418 to obtain the stable expression cells. Furthermore, EL expression level in the transfected cells were measured by fluorescence quantitative PCR, and the cell morphology was observed. Finally, CCK-8 was used to detect the effect of 584C→T variation on the cell growth of HUVECs and cell doubling time was calculated. Results： The bands positions of gel electrophoresis and restriction were in line with ex- pectations. The sequence determined was identical with it in GenBank. EL expression in transfected cells of the wild type EL and 584C T mutation type EL, detected by fluorescence quantitative PCR and mRNA electrophoresis, were significantly higher than that of empty vector. Cell morphology of the transfected cells of empty vector, wild-type EL and 584C→T EL mutant and normal HUVEC were basically the same. In accordance with it, the growth curve and cell doubling time among them were no difference. Conclusion： Stable eukaryotic expressing vector of the wild type and 584C→T variation of human endothelial lipase were successfully constructed, and the transfected cell models were established. The variation of EL had no obvious effecton the growth of HUVECs.