Electroporation-mediated In Vivo AT2R Gene Transfection Inhibits Neointimal Hyperplasia After Balloon Angioplasty In Rat Common Carotid Arteries

据世界卫生组织(WHO)统计,2010年,冠状动脉粥样硬化性心脏病(冠心病)所致死亡占总死亡人口的10%,位居第一[1]。在我国,有大约800-1000万的冠心病患者,而且随着人们生活习惯的改变,冠心病的发病越来越年轻化,一些青中年人因心肌梗死进入急诊室甚至死亡。随着经皮冠状动脉球囊成形术(PTCA)及经皮冠状动脉支架植入术(PCI)广泛应用于心绞痛或心肌梗死的治疗并取得了显著疗效,但术后半年有30%～40%的病人出现再狭窄(RS)[2,3]。应运而生的药物涂层支架虽使RS率有所下降。然而支架内RS仍然是一个棘手的问题[6]。RS病人普遍出现症状复发并需要进行再次干预治疗,其经济耗费是相当高的,也显著影响患者术后的远期疗效。PTCA或PCI时,球囊损伤动脉内皮及中层,引起血管平滑肌细胞(VSMC)表型转化,迁移,增殖及细胞外基质合成,形成新生内膜。新生内膜的形成和增殖是引起支架内再狭窄的关键因素[7]。血管平滑肌细胞过度增殖内皮细胞损伤,脱落,基底膜,致血小板聚集,与损伤的内皮细胞分泌内皮素(ET),5羟色胺(5HT),血管紧张素(Ang)等因子,引起血管收缩,同时促凝血物质如血栓素A2(TXA2),血小板源性生长因子(PDGF),二磷酸腺苷(ADP)等释放增加,使血小板黏附和聚集,形成血栓[8,9]。血管重塑形研究表明血管成形术后早期的VSMC增生往往发生在术后2周内,但内膜增厚却持续到12周,因此血管重塑在再狭窄的发生过程中起重要作用。基因治疗再狭窄是目前国内外较关注的热点,其主要策略是对靶细胞—内皮细胞进行修饰,使其表达特定基因,发挥抑制再狭窄的作用。主要的分子靶点是细胞基因,细胞周期调节基因,抗血栓形成基因,增加一氧化氮合成的基因,抑制平滑肌细胞增殖的细胞核因子,血小板衍化生长因子,抑制血管平滑肌增生的反义核酸基因。血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)受体主要有两型,即AngⅡ1型受体即AT1R和AngⅡ2型受体即AT2R。AngⅡ可通过AT1受体的激活发挥缩血管,促细胞增生迁移,促进新生内膜形成,细胞外基质沉积,促发炎症,血栓形成及氧化应激的效应。AT2受体激活则发挥舒张血管,抗增生抗血栓,诱导凋亡及产生一氧化氮NO的效应。因此对RAS进行干预,从受体水平阻断AT1 angioplasty.In vivo is the method which can directly transfer the Purpose gene into the target cells.In vivo is the most directly gene therapy.The most uesd of gene carrier is virus carrier (such as adenovirus carrier, retroviruses carrier) and the non-virus carrier (such as plasmid carrier, liposomes) carrier, however,they have many fault. The physical phenomenon of electroporation has been successfully exploited in vitro for the delivery of genes. Electriceld effects on living cells have been under investigation since the1960-1970,This first research primarily dealt with describing the phenomena of reversible and irreversible membrane breakdown in vitro.This physical phenomenon was termed electroporation. Numerous studies have demonstrated that electroporation can successf deliver the plasmidDNA into target cells.Therfore,we make the clamp shape vsscular electrode sheet which can apply with the vsscular electroporation,to study the effects of Electroporation on the AT2R gene transfected into rat carotid arteries and study the effects of AT2R gene transfer on neointimal hyperplasia in rat carotid arteries after balloon angioplasty. Methods1.Utilizing plasmid pUHD-10.3/AT2R as a template, the full length cDNA fragment of AT2R gene was amplified by polymerase chain reaction (PCR). Eukaryotic expression vector pEGFP-N2/AT2R was constructed by cloning AT2R gene into vector pEGFP-N2.2.Make the clamp shape vsscular electrode sheet which can apply with the vsscular electroporation.3.Electroporation-mediated AT2R gene transfected into rat carotid arteries after the establishment of rat carotid balloon injury restenosis model.4. The expression of AT2R in arteries were evaluated by immunohistochemistry.5. The expression of AT2R in arteries were evaluated by RT-PCR.6. The influence on neointimal of AT2R expression were evaluated by HE stainingResults1. Eukaryotic expressing vector pEGFP-N2/AT2R has been successfully constructed and confirmed identical to the sequence in database of gene bank by sequencing test.2. The clamp shape vsscular electrode can be used for in vivo Electroporation .3. The balloon catheter injury can establish an arterial restenotic model. 4. Electroporation-mediated AT2R gene delivered into injured rat carotid arteries significantly up - regulated the levels of AT2R in neointima from day7 to day 14. compared with no Electroporation-mediated group,no transfection group and GFP transfection group,the Electroporation-mediated gene transfer group, the levels of AT2RmRNA in Electroporation-mediated gene transfer group is significantl increase.5. The result of RT-PCR is that electroporation-mediated AT2R gene delivered into injured rat carotid arteries significantly up - regulated the levels of AT2R mRNA in neointima from day7 to day 14. At day 21, compared with no Electroporation-mediated group,no transfection group and GFP transfection group., the levels of AT2RmRNA in Electroporation-mediated gene transfer group is significantl increase.6. AT2R transfection significantly reduced Neointima/media area ratio (I/M) significantly(0.76±0.08),(1.39±0.08),(1.32±0.10),P<0.01).Conclusions1. The results of this study provide evidence that in vivo electroporation is an effective means for introducing naked AT2R DNA into the blood vessel wall ;2. Gene transfer of AT2R in vessel wall may effectively inhibit VSMC proliferation and neointimal hyperplasia in the rat carotid arteries after balloon angioplasty.