摘要
目的探讨局部干扰磷脂酰肌醇3激酶(P13K)p110β亚单位对移植血管新生内膜增生的作用。方法大鼠颈静脉分支-颈动脉间置模型,通过 Pluronic F-127-质粒缓释系统在血管吻合完成后局部 RNA 干扰。分6组:第1组:25%Pluronic F-127组,第2组:pU6-Pik3cb-shRNA-1组,第3组:pU6-Pik3cb-shRNA-2组,第4组:1/2(shRNA 1+shRNA 2)组,第5组:pGenesil-1质粒错配shRNA 组,第6组:渥曼青霉素阳性对照组。实验时分别将200μl含有50μg shRNA 质粒,或50μg渥曼青霉素,或空白 Pluronic F-127凝胶均匀地涂在移植桥血管的周围。每组30只 SD 大鼠,分别于1、3、7、14、28 d 取材苏木精-伊红染色观察内膜厚度;术后3 d 进行荧光定量 PCR 和 Western 印迹法测定 P13K p110β亚单位 mRNA 和其下游效应分子磷酸化 Akt 和 mTOR。结果 Pluronic F-127质粒缓释系统平滑肌细胞转染效率为60%,内皮细胞达90%以上,pU6-Pik3cb-shRNA 和渥曼青霉素转染后 Pik3cb mRNA 和其下游效应分子磷酸化 Akt 和 mTOR 下调,移植血管内膜增生减轻。结论靶向Pik3cb 的 shRNA 下调 P13K-Akt-mTOR 信号通路,减轻移植血管新生内膜增生。
Objective To investigate whether knockdown Pik3cb p110β subunit by shRNA in autologous vein grafts can reduce intimal hyperplasia. Methods 180 adult SD rats underwent carotid artery bypass graft surgery by using the autologous branch of jugular vein, and they were randomly divided into 6 equal groups： Group A （with the jugular vein grafts treated with 25% Pluronic F-127 only）, Group B （ with the graft treated with the plasmid encoding shRNA targeting Pik3cb p110β subunit, pU6-Pik3cb-shRNA-1 ）, Group C （with the graft treated with the plasmid encoding shRNA targeting Pik3cb p110β subunit, pU6- Pik3cb-shRNA-2）, Group D （with the graft treated with the half pU6-Pik3cb-shRNA-1 and pU6-Pik3cbshRNA-2）, and Group E （with the graft treated with the pGenesil-1 scramble shRNA）, and Group F （with the jugular vein grafts treated with wortmannin）. Specimens of jugular vein graft were harvested 1, 3, 7, 14, and 28 days after surgery to assess the neointimal hyperplasia. Another 18 rats were randomly divided into 6 equal groups as mentioned above to be used in a parallel experiment： 72 h after surgery specimens of jugular vein graft were harvested to undergo fluorescence quantitative real-time PCR and Western blotting to detect the mRNA expression of P13K p110β subunit and the protein expression of phophorylated Aktphospho-Akt （ Thr 308 ） and phospho-Akt （ Ser473 ）-, and mTOR （ Ser 2448 ）. And another 9 rats received jugular vein grafts treated with the pGenesil-1 scramble shRNA, and on the postoperative days 1, 2, and 3 respectively 3 rats were killed to undergo fluorescence staining to detect the transfection efficacy. Results The transfection rate of the plasmid pGenesil-1 was 60% in the vascular smooth muscle cells and 90% in the endothelial cells. The thickness of tunica intima 28 days after the surgery of the pU6-Pik3cb-shRNA-1, pU6-Pik3cb-shRNA-2, 1/2 （ shRNA1 ＋ shRNA2 ） , and wortmannin groups were （ 34.6± 2.7 ） μm, （39.4 ± 2.5 ）μm, （36.7 ± 2.9） μm, and （40.6± 3.1 ） μm respectively, all significantly lower than that of the control group （61.8 ±4.3 μm,P 〈0.05）. The expression levels of phospho-Akt （Thr 308） , phospho-Akt （Ser 473 ） , and mTOR of the shRNA intervention and wortmannin groups were all downregulated. Conclusion Knockdown of Pik3cb in interposition carotid artery branch of jugular vein grafts reduces intimal hyperplasia with the possible mechanism of downregulation of phosphatidylinositol 3-kinase signaling through Akt, with resultant decreases in VASC growth and survival. Modulation of the phosphatidylinositol 3-kinase pathway through knockdown Pik3cb may represent a novel therapy to prevent vein graft intimal hyperplasia after bypass grafting.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第24期1713-1716,共4页
National Medical Journal of China
基金
山西省科技攻关基金(042025-1)
山西省回国留学人员科研基金(74-2003)
关键词
移植物闭塞
血管
血管内膜
增生
Graft occlusion, vascular
Tunica intima
Hyperplasia
作者简介
邓勇志,现在山西医科大学第二医院心胸外科
柯俊,现在广东省心血管疾病研究所
