腺病毒介导血管平滑肌细胞转染表达血管紧张素Ⅱ2型受体的研究

The Study of the Transfer and Expression of Angiotensin Ⅱ Type 2 Receptor Gene in Rat Vascular Smooth Muscle Cells Mdiated by Recombinant Adenovirus Vector

目的 体外培养大鼠血管平滑肌细胞 (VSMC)并以腺病毒介导转染表达血管紧张素Ⅱ (AngⅡ ) 2型受体 (AT2 R)。方法 取大鼠主动脉血管 ,以常规组织块贴壁法培养VSMC ;用同源重组方法构建带AT2 R基因的复制缺陷型腺病毒载体(AdCMV AT2 R) ,并加以鉴定、扩增 ,以制取高滴度AdCMV AT2 R转染液 ,体外转染VSMC ;用RT PCR方法检测AT2 RmRNA表达 ,免疫组织化学法及蛋白免疫印迹法检测AT2 R蛋白表达 ,流式细胞仪检测AT2 R表达率 ,同时检测AT1R的表达变化。结果 构建的AdCMV AT2 R体外转染培养VSMC表达率为89.5 1%。以免疫组化、免疫印迹和RT PCR检测AT2 R表明 ,转染后AT2 R表达明显增强 ,并随表达时间延长而增加 ,48小时达峰值。AT2 R转染表达不影响AT1R表达。结论 腺病毒载体可较高效率介导AT2 R在体外培养的VSMC转染表达 ,AT1R表达不受其影响。转染表达AT2 R的VSMC可作为研究AngⅡ对其增殖。

Objective\ To constructed the cell model transferred angiotensin Ⅱ (AngⅡ) type 2 receptor (AT\-2R) in vascular smooth muscle cells(VSMC) Method\ The VSMCs, isolated from the aorta of rat, were cultured by routine method. Recombinant adenoviral vector, AdCMV\|AT\-2R, containing rat AT\-2 receptor gene was constructed by homologous recombination, and then it was used to transfer AT\-2 receptor gene to VSMC in vitro. The rate of AT\-2R expression in VSMC was analysed by flow cytometry. The expression of mRNA, protein were detected by RT\|PCR, Western blot and immunohistochemistry respectively. The angiotensin Ⅱ (AngⅡ) type 1 receptor (AT\-1R) was determined as well. Result\ The expression rate of AT\-2R in VSMC was increased significantly after transferred by AdCMV\|AT\-2R with time, and the peak value detected by flow cytometry was about 89.51% at 48 hours. RT\|PCR, Western blot and immunohistochemistry showed that the expression of AT\-2R mRNA and protein were increased obviously in transferred VSMC. There were no significantly change of AT\-1R expression during AT\-2R expression. Conclusion\ Our study indicates that AdCMV\|AT\-2R did generate high level expression of AT\-2 receptor and its expression did not affect AT\-1R expression in cultured VSMC. The VSMCs transferred AT\-2R gene may be used as a good model to study the effect of AT\-2R on their biological action such as proliferation, migration and apoptosis.