纳米粒子介导血管紧张素II的1型受体相关蛋白对大鼠血管内膜增生的影响

There is no effective countermeasures to treat the neointimal formation after percutaneous transluminal angioplasty or vascular bypass operation currently. Convenient and effective methods or medication are needed urgently to solve this difficult problem clinically. Our preliminary experiments have shown that Angiotensin II receptor type 1 associated proteins (ATRAP) can inhibit vascular smooth muscle cells (VSMC) proliferation and the neointimal hyperplasia of blood vessels. In this project, PLGA and PVA containing ATRAP gene plasmid are applied to make nanoscale particle mixtures. Using the model of carotid injury of rats in vivo and VSMC of rats in vivro via nanoparticles as a gene transfection vector, and taking VSMC apoptosis, the changes of vascular inflammatory cytokines and the effect on neointimal formation as observational targets to evaluate the efficiency of nanoparticle as a gene transfection vector, study the effect of ATRAP mediated by nanoparticles on VSMC apoptosis , inhibition of neointimal formation and its molecular mechanism, manufacture the medical fibrin glue or suspended emulsion containing ATRAP gene mediated by nanoparticles, which can be applied either directly deposit on the injured vascular adventitia during operation or through the endovascular intervention to solve the worldwide puzzle which the intimal hyperplasia after vascular surgery.

目前国内外对经皮血管成型及血管旁路术后血管内膜增生均无有效对策，临床上迫切需要方便有效的方法或药物来解决该临床难题。我们的前期工作已证明了血管紧张素II的1型受体相关蛋白（ATRAP）可以抑制血管平滑肌细胞（VSMC）增殖及血管内膜的增生。本项目应用聚乳酸聚乙醇酸共聚物（PLGA）和聚乙烯醇（PVA）包载ATRAP基因质粒，制备纳米级粒子混合物，在大鼠离体VSMC及大鼠颈血管损伤模型上，通过纳米粒子介导的基因转染的方法，以VSMC凋亡、血管炎性因子的变化及对血管内膜增生的影响为观测指标，评价纳米粒子携带ATRAP基因质粒转染的效率；论证纳米粒子介导的ATRAP在VSMC凋亡及血管内膜增生抑制上的作用及其细胞分子机制。制备包含在医用蛋白胶或悬浮乳液内的纳米粒子介导的ATRAP基因制剂，使其可以直接在术中涂于受损血管外膜或通过腔内介入的方法到达受损血管，解决目前血管外科术后内膜增生的世界难题。

血管紧张素Ⅱ1型受体相关蛋白（ATRAP）通过负调控AT1R的作用参与调节血管平滑肌细胞（VSMC）的增生与凋亡。本课题通过构建ATRAP过表达的腺病毒，结合运用流式细胞术、TUNEL、荧光定量PCR、蛋白质生物化学、免疫荧光等方法与技术，对ATRAP调控VSMC的凋亡并抑制颈动脉内膜增生的作用及相关分子机制进行了初步研究。首先通过构建带FLAG标签的ATRAP过表达的腺病毒及对照病毒，转染体外培养的VSMC，观察到ATRAP促进VSMC凋亡，且不依赖于AngⅡ的存在；而应用PTEN抑制剂可以逆转ATRAP对VSMC的促凋亡作用；通过RT-PCR结果显示,Adv.ATRAP组相比对照组（Adv.GFP）促凋亡基因Bax mRNA表达量明显增高，而抑制凋亡的Bcl-2 mRNA表达量降低；western blot结果也显示Adv.ATRAP组相比对照组（Adv.GFP），cleaved-caspase 8、cleaved-caspase3、Bax表达增加，而Bcl-2蛋白表达量减低，cleaved-caspase 9表达量没有明显差异，也证实ATRAP过表达可诱导VSMC凋亡，且主要是通过死亡受体途径（外源性途径）；同时Adv.ATRAP组相比对照组（Adv.GFP）组，血管紧张素Ⅱ诱导的Akt磷酸化受到抑制，而PTEN抑制剂可很大程度上逆转这种抑制作用。用腺病毒转染球囊损伤的大鼠颈总动脉，术后7天、14天可以观察到Adv.ATRAP转染组内膜增生明显受抑制，且内膜中膜区细胞凋亡明显增加。综合上述结果，本研究表明ATRAP可以通过调控PI3K/Akt信号通路促进VSMC的凋亡并抑制颈动脉的内膜增生，从而为临床治疗颈动脉再狭窄提供了新的治疗方向与理论基础。项目资助发表核心论文2篇。培养博士生1名，硕士生2名。项目投入经费55.0万元，支出54.7124万元，余额2876元，各项支出基本与预算相符。