下调大鼠血管平滑肌细胞血管紧张素转换酶的表达

摘要

目的 构建腺病毒载体使其携带血管紧张素转换酶(ACE)基因短发夹RNA(shRNA),观察选择性下调大鼠血管平滑肌细胞ACE表达.方法 从之前构建的真核表达载体p-ACE-shRNA中扩增ACE-shRNA片段,采用RT-PCR法并克隆进穿梭质粒pDC316中,将构建好的pDC316-ACE-shRNA穿梭质粒载体和pBHGlox-E1,3Cre骨架病毒共转染293细胞,进行病毒颗粒包装重组、滴度测定和纯化.随后进行原代培养大鼠血管平滑肌细胞转染,通过实时荧光定量PCR分别在转柒前及转染后24h、48h、72 h检测ACE mRNA的表达.结果 经PCR检测证实携带ACE-shRNA重组腺病毒载体构建成功并制备出了高滴度重组病毒,大鼠血管平滑肌细胞被转染后24 h,ACE mRNA表达无明显变化;被转染后48h,ACE mPNA表达显著降低,差异有统计学意义(P＜0.05);被转染后72h时ACE mRNA表达更低.结论 本实验成功构建重组腺病毒载体其携带ACE-shRNA片段,同时证实shRNA可选择性下调原代培养大鼠血管平滑肌细胞上ACE表达,可能为心血管病基因治疗提供新思路.%Objective To construct adenovirus vector carrying the shRNA of rat angiotensin-converting enzyme (ACE).To selectively knock down the expression of angiotensin-converting enzyme (ACE) in rat vascular smooth muscle cells by RNA interference.Methods The rat ACE-shRNA segments was obtained from plasmid p-ACE-shRNA which was constructed at an earlier date by RT-PCR and then was cloned into the shuttle plasmid pDC316 to form the pDC316-EGFP-ACE-shRNA vector.The pDC316-EGFP-ACE-shRNA plasmid was cotransfected with genomic plasmid pBHGlox-E1,3Cre into 293 cells to package the recombinant adenovirus.The recombinant adenovirus was transfected into the primary cultured rat vascular smooth muscle cells.ACE mRNA expression were analyzed from rat vascular smooth muscle cells before transfection and 24,48,72 hours after transfection by real-time fluorescence quantitative PCR.Results Recombinant adenoviral vector Ad-EGFP-ACE-shRNA was constructed successfully,which was confirmed by restriction enzyme digestion,PCR and GFP expression.Expression of ACE mRNA in rat vascular smooth muscle cells were significantly reduced 48 hours after Ad-EGFP-ACE-shRNA transfection,and was nearly undetectable after 72 hours.Conclusion The recombinant adenoviral vector carrying ACE-shRNA is successfully constructed.RNA interference can knockdown ACE expression in cultured rat vascular smooth muscle cells,which maybe provide.a new gene therapy idea for cardiovascular diseases.