反义AT1R腺病毒穿梭质粒的构建及其在血管平滑肌细胞中的表达

Construction of adenoviral shuttle plasmid containing the antisense cDNA of human AT1R and its expression in vascular smooth muscle cells

目的 构建含人血管紧张素Ⅱ 1型受体 (AT1R)反义cDNA(ahAT1 )的腺病毒穿梭质粒 ,观察其在血管平滑肌细胞 (VSMC)中的表达 ,为ahAT1抗高血压、PTCA术后再狭窄及肺动脉高压的研究奠定基础。方法 用定向克隆法将人AT1RcDNA反向插入到穿梭质粒pACCMVPLPA的CMV启动子之下 ,即获得重组腺病毒穿梭质粒pAd/CMV .ahAT1。以脂质体包裹质粒并转染VSMC ,用免疫细胞化学方法和图像分析鉴定此质粒对VSMC表达AT1R的影响。结果 AT1RcDNA成功地反向插入到pACCMVPLPA载体 ,与对照组相比 ,pAd/CMV .ahAT1明显抑制了VSMC的AT1R表达 (P <0 0 5 )。结论 成功构建了携带反义人AT1RcDNA的腺病毒穿梭质粒 。

Objective To construct adenoviral shuttle plasmid containing the antisense cDNA of human AT1R (hAT1R),and to determine its expression in vascular smooth muscle cells (VSMC) for future gene therapy of systemic hypertension,pulmonary hypertension and restenosis after percutaneous transluminal coronary angioplasty(PTCA).Methods The hAT1R cDNA was extracted from pRcCMVhAT1R with Hind Ⅲ and Xba Ⅰ and then inserted into the E1 deleted expression plasmid pACCMVPLPA shuttle vector with antisense orientation,called pAd/CMV.hAT1R.The plasmids (pAd/CMV.hAT1R or control plasmid called pAd/CMV.LacZ) were mixed with lipofectamine,and then the plasmids were encapsulated to lipofectamine and transfected into VSMC ex vivo.After 2 days of the transfection,the level of AT1R protein was detected by immunohistochemical method.Results AT1R cDNA was successfully inserted into the shuttle vector pACCMVPLPA.pAd/CMV.hAT1R was confirmed by Hind Ⅲ and Xba I digestion.The expression of AT1R protein markedly decreased in pAd/CMV.hAT1R group than that in control group( P <0.05).Conclusions pAd/CMV.hAT1R containing the antisense hAT1R cDNA sequence was successfully constructed and effectively expressed in vascular smooth muscle cells.