平滑肌22α启动子/增强子靶向调控哺乳动物雷帕霉素靶蛋白信号通路对移植血管平滑肌细胞增殖的影响  

Modification of mammalian target of rapamycin signaling pathway under the driving of specific SM22α promoter/enhancer influences the vein graft vascular smooth muscle cells hyperplasia

作　　者：陈志强[1] 王帅[1] 邓勇志[1] 郑志发[1] 

机构地区：[1]山西省心血管病医院(研究所)山西医科大学附属心血管病医院心血管外科,太原030024

出　　处：《中华实验外科杂志》2015年第2期246-249,共4页Chinese Journal of Experimental Surgery

基　　金：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资助项目（11014926）

摘　　要：目的 采用血管平滑肌细胞（SM）特异性SM22α启动子/平滑肌肌球重链蛋白（SMHC）增强子,构建靶向大鼠哺乳动物雷帕霉素靶蛋白（mTOR）基因的干扰RNA真核表达载体,研究其对血管平滑肌细胞（VSMC）增殖及移植血管新生内膜增生的影响.方法 构建大鼠特异性SM22α真核表达载体SM22α-p/e-mTOR-短发卡RNA（shRNA）,建立大鼠颈静脉-动脉移植模型,在血管吻合完成后通过Pluronic F-127质粒缓释系统对血管内平滑肌增殖进行局部RNA干扰.实验分5组,每组20只SD大鼠.A组：对照组（25％ Pluronic F-127组）;B组：SM22α组（SM22α-p/e-mTOR-shRNA）;C组：巨细胞病毒（CMV）组（CMV-p/e-mTOR-shRNA）;D组：阴性对照组（空质粒pGenesil-10）;E组：阳性对照组;其中,B组和C组统称实验组.根据实验分组的不同,将含有50 μg shRNA质粒,或50 μg渥曼青霉素（wortmannin）凝胶,或单纯200μl 25％ Pluronic F-127凝胶均匀涂抹在移植静脉周围,分别于术后1、3、7、14 d获取移植血管.苏木素-伊红（HE）染色观察新生内膜厚度;免疫组织化学检测平滑肌肌动蛋白（α-SM-actin）、增殖细胞核抗原（PCNA）、磷酸化-mTOR （Ser2448）,原位末端转移酶标记技术（TUNEL）检测细胞凋亡,评估mTOR信号通路的变化以及平滑肌细胞的增殖.结果 术后7d各组新生内膜厚度差异有统计学意义（P＜0.05）;术后14 d,A组新生内膜较术后1d增厚12.4倍;B、C、D、E组分别增厚9.7、4.8、7.6、2.6倍.术后14d,各实验组和阳性对照组移植静脉α-SM-actin表达阳性面积均明显低于对照组（P＜0.05）,PCNA阳性表达面积均低于对照组（P＜0.05）,磷酸化-mTOR（Ser2448）阳性表达面积均低于对照组（P＜0.05）;实验组、对照组、阴性对照组术后移植静脉凋亡细胞阳性面积比较差异无统计学意义（P＞0.05）,阳性对照组凋亡面积明显高于其他组（P＜0.05）.结论 SM22α-p/e-mTOR-shRNA可通过抑制血管Objective Construct targeting rat mammalian target of rapamycin （mTOR） eukaryotic expression vector by the specific of vascular smooth muscle cell （VSMC） SM22α promoter/smooth-muscle myosin heavy chain （SMHC） enhancer,and explore the mechanism of VSMC proliferation and vein graft neointimal hyperplasia.Methods Rat VSMC specificity eukaryotic expression vector of SM22α-p/e-mTOR-shRNA was constructed,and rat vein-to-artery interposition model also constructed using the autologous branch of jugular vein.Jugular vein grafts were treated with 25% Pluronic F-127 only （group A,n =20）,plasmid encoding short hair-pin RNA （shRNA） targeting mammalian target of rapamycin （mTOR） （SM22oα-p/e-mTOR-shRNA and CMV p/e-mTOR-shRNA,groups B and C,n =20,respectively）,pGenesil-10 （group D,n =20）,or wortmannin （group E,n =20）.Specimens were harvested at 1,3,7 and 14 days after surgery to assess jugular vein graft neointimal hyperplasia through hematoxylin and eosin （HE） staining.Immunohistochemical staining were performed with primary antibodies of phosphor-mTOR （Ser2448）,α-Smooth muscle actin,proliferation cell nuclear antigen （PCNA）,as well as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） method to evaluate the antiproliferative effects of shRNA.Results Seven days after surgery,there were significant differences of neointimal thickness in the positive control group and the plasmid transfection groups compared with the control group （P 〈 0.05） ; 14 days after surgery,the intimal thickness of control group were significantly increased when compared with the positive control group and the plasmid transfection groups （P 〈 0.05）.Fourteen days after surgery,the neointimal thicken 12.4 times compared with the one day in the group A; groups B,C,D and E were 9.7 times,4.8 times,7.6 times and 2.6 times,respectively; Vein graft α-SM-actin positive expression area in positive control group and in the plasmid transfection groups were significantly

关 键 词：新生内膜增生 哺乳动物雷帕霉素靶蛋白 血管平滑肌细胞 SM22α启动子/增强子 

分 类 号：R654.3[医药卫生—外科学]