【摘要】 目的研究雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)及其底物——核糖体蛋白S6激酶(p70s6k)、真核细胞启动子4E结合蛋白1(4E-BP1)在自体移植静脉中表达的动态变化规律,探讨其与内膜增生的关系及意义。方法Wistar大鼠64只,建立自体静脉移植模型,随机分为8组,分别在移植后6 h,1、3 d,1、2、4、6、8周切取移植静脉。逆转录-聚合酶链反应(RT-PCR)联合原位杂交方法检测移植血管中mTOR及其底物p70s6k和4E-BP1的mRNA表达,Western蛋白印迹或免疫组化方法检测mTOR、p70s6k和4E-BPI的蛋白产物表达,同时检测增殖细胞核抗原(PCNA)的表达。结果静脉移植1～3 d即出现mrFOR和p70s6k的mRNA表达增强,3 d～2周达到高峰,与其他组比较差异有统计学意义(P<0．01),6～8周逐渐恢复正常;而4E-BP1的mRNA表达在移植后1 d开始降低,1周表达最低,之后表达开始增强,4～6周为表达高峰,与其他组比较差异有统计学意义(P<0．01)。Western蛋白印迹提示mTOR和p70s6k蛋白产物在移植后1周表达明显增加,2～4周达到高峰,8周恢复至正常水平;而4E-BP1蛋白产物在移植后1周表达最少,4-6周增加达到高峰,8周仍维持一定的表达量。原位杂交及免疫组化结果提示阳性细胞多定位于移植静脉新生内膜和中膜血管平滑肌细胞,mTOR及其底物与PCNA分布基本一致。结论mTOR信号通路在血管移植后即被激活并与内膜增生关系密切,可能成为防治移植血管狭窄、闭塞的有效靶点。更多还原

【Abstract】 Objective To investigate the expression of mammalian target of rapamycin (mTOR) and its substrates including p70s6k and 4E-BP1 in autogenous vein graft. Methods Autogenous vein graft model was established in 64 Wistar rats by transplanting the right common jugular vein to infrarenal abdominal aorta. Vein graft samples were harvested 6 hours, 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks after surgery. The mRNA expression of mTOR, p70s6k and 4E-BP1 were measured by RT-PCR and in situ hybridization. Western blot and immunohistochemistry methods also were used to detect the protein expression of mTOR,p70s6k and 4E-BP1. Proliferating cell nuclear antigen(PCNA) was also detected at the same time. Results The mRNA expression of mTOR and p70s6k increased soon after vein graft transplanting, rose quickly and reached the peak 3 days to 2 weeks after surgery, which recovered 6 to 8 weeks after surgery. The expression of 4E-BP1 mRNA decreased soon after surgery and reached the lowest at 1 week, then rose to the peak 4 to 6 weeks after transplantation. Protein expression of mTOR and p70s6k reached the peak 2 to 4 weeks and recovered to normal level 8 weeks after surgery, but the expression of 4E-BP1 decreased to the lowest during 1 to 2 weeks and reached the peak 4 to 6 weeks after transplanting. The positive cells mostly located in vascular smooth muscle cell ( VSMC) just like PCNA. Conclusions The expression of mTOR and its substrates were activated in vein graft soon after transplantation, which means that mTOR and its substrates might become new targets for the prevention and therapy of stenosis or obliteration after vein graft transplanting.更多还原