血管紧张素Ⅱ2型受体重组腺病毒的扩增及其在INS-1细胞中的转染

Amplification of recombinant adenovirus vector carrying rat angiotensin Ⅱ type 2 receptor and transfection in INS-1 cells

目的利用人胚肾(HEK)293A细胞株扩增含有大鼠血管紧张素Ⅱ2型受体(AT2R)基因的重组腺病毒,并在大鼠胰岛素瘤细胞INS-1细胞株中进行转染,构建AT2R高表达的胰岛β细胞模型。方法利用293A细胞株扩增重组腺病毒Ad-G-AT2R-EGFP和对照病毒Ad-CMV-EGFP,并测定病毒滴度。在INS-1细胞中瞬时转染后利用实时PCR、Western印迹以及免疫荧光和激光共聚焦技术检测细胞中AT2R与AT1R的表达。结果扩增后得到重组腺病毒Ad-G-AT2R-EGFP和Ad-CMV-EGFP的滴度分别为9×109和8×109pfu/ml。在INS-1细胞中转染Ad-GAT2R-EGFP后,AT2R mRNA的表达随病毒感染复数(multiplicity of infection,MOI)值的增加呈剂量依赖性增加。当MOI值为10时,AT2R mRNA的表达达到峰值(P<0.05),同时AT2R蛋白的表达明显高于转染了Ad-CMV-EGFP的细胞和未转染细胞,但AT1R的表达无显著变化。结论成功扩增并得到高滴度且携带有AT2R基因的重组腺病毒载体,并将其转染入INS-1细胞中构建出AT2R高表达的胰岛β细胞模型,为下一步探讨AT2R在胰岛β细胞中的作用奠定了基础。

Objective To amplify the recombinant adenovirus vector carrying rat angiotensin Ⅱ type 2 receptor（AT2R） gene using human embryonic kidney（HEK） 293 A cell lines and to construct a pancreatic islet β cell model overexpressing AT2 R by transfecting the adenovirus vector into rat insulinoma（INS-1） cell lines.Methods Recombinant adenovirus vector Ad-G-AT2R-EGFP and control vector Ad-CMV-EGFP were amplified with HEK 293 A cells and the titer of the adenovirus was detected.After both adenovirus vectors were transfected into INS-1 cells,AT2 R and angiotensin Ⅱtype 1 receptor（AT1R） gene expressions were tested using real-time PCR,Western blotting,immunofluorescence staining and confocal laser-scanning microscopy.Results The titer of amplified Ad-G-AT2R-EGFP and Ad-CMV-EGFP was respectively 9 × 109 pfu / ml and 8 × 10^9 pfu / ml.Transfection of Ad-G-AT2R-EGFP into INS-1 cells induced an increase in AT2 R mRNA expression in a dose-dependent manner,and significantly increased AT2 R mRNA and protein expression compared with Ad-CMV-EGFP-or mock-transfection.Conclusion The recombinant adenoviral vector carrying AT2 R gene is successfully amplified and an INS-1 cell model overexpressing AT2 R is constructed by transient transfection,which can contribute to further study of the role of AT2 R in pancreatic islet β cells.