BTEB2反义基因转染对大鼠颈动脉球囊损伤后内皮修复的影响

Effects of BTEB2 antisense RNA on reendothelialization after rat carotid artery injury

目的构建反义基本转录元件结合蛋白2(BTEB2)重组腺病毒载体,并研究BTEB2反义RNA对动脉损伤后内皮修复的影响。方法通过RTPCR从培养的大鼠血管平滑肌细胞(VSMC)中制备BTEB2cDNA,将其反向克隆至腺病毒载体,构建反义BTEB2重组腺病毒;用重组腺病毒局部转染球囊损伤的大鼠颈动脉,观察反义BTEB2基因转染对BTEB2蛋白表达及损伤内皮修复的影响。结果构建的BTEB2反义重组腺病毒经鉴定正确,其滴度为5×1010pfumL;反义BTEB2重组腺病毒转染可显著抑制BTEB2蛋白表达,明显促进内皮的修复,改善损伤动脉的内皮依赖性舒张功能。结论成功构建了反义BTEB2重组腺病毒载体。BTEB2反义基因转染可显著促进大鼠颈动脉球囊损伤后的内皮修复。

Objective To construct recombinant adenovirus vector of Antisense BTEB2 （Ad. ASBTEB2） and study the effects of BTEB2 antisense RNA on reendothelialization after rat carotid artery injury. Methods BTEB2 cDNA was prepared by RT-PCR technique and was subcloned reversedly into adenovirus vector to construct Ad. ASBTEB2. The PCR technique was used to detect target gene fragment and adenovirus genomic characteristic fragment. Antisense BTEB2 gene was transduced into rat carotid arteries with Ad. ASBTEB2 after the establishment of rat carotid balloon injury models. The BTEB2 expression was detected by immunohistochemistry. The reendothelialization of injured arteries was evaluated by Evans blue staining. Acetylcholine （ACh） was used to assess the endothelium-dependent vasorelaxation of isolated arterial rings. Results The recombinant adenovirus was proved correct by PCR. Ad. ASBTEB2 delivered into injured rat carotid arteries reduced BTEB2 protein expression significantly from day 7 to 21 after injury. By 21 days after injury, Evans blue dye staining disclosed more obvious reendothelialization of the Ad, ASBTEB2-treated balloon-injured artery compared with the Ad, LacZtreated injured vessel, Endothelium-dependent vasorelaxation by acetylcholine was more pronounced in the Ad. ASBTEB2-treated group than in the Ad, LacZ-treated group and the un-treated group. Conclusion Local endovascular delivery of Ad. ASBTEB2 at the injury site is able to block the BTEB2 protein overexpression, and improve the degree of reendothelialization sufficiently to restore endothelium-dependent relaxant function to the injured carotid arteries.