下调大鼠血管内皮细胞血管紧张素转换酶的实验研究

Experimental Study on Knockdowning of Angiotensin-Converting Enzyme in Rat Vascular Endothelial Cells

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),通过RNA干扰技术选择性地下调大鼠血管内皮细胞(ECs)ACE表达。方法采用RT-PCR法从之前构建的真核表达载体p-ACE-shRNA中扩增ACE-shRNA片段,扩增之后将ACE-shRNA片段克隆进pDC316穿梭质粒,再将293细胞被构建好的pDC316-ACE-shRNA穿梭质粒载体和pBHGlox-E1,3Cre骨架病毒转染,进行病毒颗粒包装重组并进行滴度测定和纯化。随后进行原代培养大鼠血管ECs转染,通过实时荧光定量PCR法分别在转染前及转染后24h、48h、72h通过实时荧光定量PCR法来检测ACE mRNA的表达。结果高滴度的重组病毒被制备完成,携带ACE-shRNA的重组腺病毒载体经PCR检测、限制性内切酶和GFP表达证实构建成功,被转染24h时大鼠血管ECsACE mRNA表达无统计学意义变化;但被转染48h时,ECsACE mRNA表达差异有统计学意义(P<0.05);被转染后72h时表达更低。结论实验成功构建重组腺病毒载体并携带ACE-shRNA片段,同时证实shRNA可选择性下调原代培养大鼠血管ECs上ACE表达,可能为心血管病基因治疗和应用提供新思路。

Objective The adenovirus vector was constructed to carry the short hairpin RNA （shRNA） of angiotensin- converting en -zyme （ACE）,and the ACE expression was selectively knockdown in rat vascular endothelia1 cells （ECs） by RNA interference.Methods The segments ACE shRNA was obtained from plasmid p -ACE -shRNA that was constructed at an earlier date,and then wascloned into the pDC316 shuttle plasmid to form the vector pDC316- EGFP -ACE- shRNA by RT PCR.To package the recombinantadenovirus,the plasmid pDC316 -EGFP -ACE- shRNA was cotransfected with pBHGlox E1,3Cre genomic plasmid into 293 cells.The recombinant adenovirus was transfected into the primary cultured rat vascular ECs.ACE -mRNA expression were analyzed fromrat vascular ECs before transfection and 24 hours,48 hours,72 hours after transfection by real time fluorescence quantitative PCR.Results Recombinant adenoviral vector Ad- EGFP- ACE -shRNA was constructed successfully that was confirmed by restrictionenzyme digestion,PCR and GFP expression.Expression of ACE mRNA in rat vascular ECs were significantly reduced 48 hours afterAd -EGFP- ACE -shRNA transfection,and was nearly undetectable after 72 hours.Conclusion The recombinant adenoviral vectorwhich carry ACE -shRNA is successfully constructed.RNA interference can knockdown ACE expression in cultured ECs,which maybeprovide a new gene therapy idea for cardiovascular diseases.