SM22α启动子／增强子基因调控磷酸肌醇3激酶信号通路对移植血管新生内膜增生的影响  

作　　者：徐俊文 邓勇志 王倩 陈丽 杨雪峰 

机构地区：[1]山西省心血管病医院研究所心血管外科,太原030024

出　　处：《中华实验外科杂志》2013年第5期917-920,共4页Chinese Journal of Experimental Surgery

基　　金：基金项目：山西省留学回国人员科研资助项目（2011-107）;太原市技术创新与人才扶持计划人才扶持专项（11014926）

摘　　要：目的采用组织特异性启动子SM22α，构建靶向大鼠磷脂酰肌醇3激酶β亚单位（Pik3cb）的短发仁RNA（shRNA）真核表达载体，观察其对静脉移植血管血栓形成和新生内膜增生的影响。方法建立大鼠颈静脉-动脉移植模型，通过Pluronic F-127质粒缓释系统在血管吻合完成后局部喷涂凝胶进行RNA干扰。实验分4组，每组30只SD大鼠，A组：对照组，空白Pluronic F-127凝胶；B组：SM22a组，含特异性SM22a启动子质粒的凝胶；C组：巨细胞病毒（CMV）组，含非特异性CMV启动子质粒的凝胶；D组：Wortmannin阳性对照组。分别于术后1、3、7、14、28d截取移植血管，苏木素-伊红（HE）染色观察新生内膜厚度；免疫组织化学检测磷酸化哺乳动物雷帕霉素靶蛋白（mTOR，Ser2448），原位末端转移酶标记技术（TUNEL）检测细胞凋亡。另设一平行实验组（12只SD大鼠），按上述方法分为4组，于术后3d取材，实时荧光定量逆转录-聚合酶链反应（VQ—PCR）测定Pik3cb mRNA的相对表达量。结果大体观察移植血管通畅率B组最高（86．7％），与对照组比较差异有统计学意义（P〈0．01）；Pik3cb mRNA相对表达量B、C、D组分别下降了73．3％、81．2％、68．4％，与对照组比较差异均有统计学意义（P〈0．01）；术后1、3、7、14、28d，B组与A组比较，磷酸化mTOR（Ser2448）阳性面积均显著降低，细胞凋亡阳性面积均显著增加，差异均有统计学意义（P〈0．05），术后7、14、28d，B组与A组比较，血管内膜厚度显著降低，差异有统计学意义（P〈0．01）。结论SM22α特异性启动子／增强子真核表达载体可通过下调血管平滑肌细胞磷酸肌醇3激酶-蛋白激酶B—mTOR（P13K—Akt—mTOR）信号通路而抑制其向内膜增殖、迁移，促进其凋亡，防止血管新生内膜增生，提高移植血管通畅率。Objective To construct rat phosphatidylinositol 3-kinase,catalytic,beta polypeptide (Pik3eb) shRNA eukaryon plasmid expression vector using SM22α specific promoter,and study its effect on thrombosis of vein grafts and neointimal byperplasia.Methods SM22α specific promoter/enhancer and cytomegalovirus (CMV) shRNA of rat Pik3cb were designed and syntbesized according to the sequence of Pik3cb in the Gene Bank,then they were annealed to form double strands and cloned into pGenesil-10,named SMHCe/SM22αp pGcnesil-10-Pik3cb-shRNA and CMV-pGenesil-10-Pik3cb-shRNA,respectively.Modified rat vein-to-artery interposition models using the autologous branch of jugular vein were constructed.The transplanted jugular vein grafts were then treated with either 25％ Pluronic F-127 only (group A,n =30),plasmid encoding shRNA targeting Pik3cb p110 03 subunit (SMHCe/SM22αp-pGenesil-10-Pik3cb-shRNA or CMV-pGenesil-10-Pik3cb-shRNA.groups B and C,n =30,respectively),or wortmannin (group D,n =30).Specimens were harested at 1st,3M,7th,14th and 28th day post-operation to assess the severity of jugular vein graft neointimal hyperplasia.Immunohistochemical staining was performed with primary antibodies phosphor-mTOR (Ser2448),as well as TdT-mediated dUTP nick end labeling (TUNEL) method to evaluate the antiproliferative effects of shRNA.Furthermore,the animals in the control groups (n =18,3 for each group) receiving the same treatment as above were sacriticed on the postoperative day 3 for assays to evaluate relative amount expression of Pik3cb mRNA.Results Rat vein-to-artery interposition models using the autologous branch of jugular vein were successfully constructed.The patency rate of vein graft in group B was 86.7％,which was significantly higher than in group A (P ＜0.01).The Pik3cb mRNA relative expression in groups B,C and D was significantly decreased by 73.3％,81.2％ and 68.4％,as compared with group A (P＜0.01).Vein graft phosphor mTOR (Ser2448) positive expression area in group B was reduced by 55.2％,56.8％,57.2％,5

关 键 词：新生内膜增生 磷脂酰肌醇3激酶 血管平滑肌细胞 SM22α启动子 

分 类 号：R65[医药卫生—外科学]