加味通幽汤对食管癌Eca-109细胞mTOR/HIF-1α通路及肿瘤缺氧相关因子的影响  

Effects of modified Tongyou Decoction on mTOR/HIF-1α pathway and tumor hypoxia inducible factors in Eca-109 cells of esophageal cancer

作　　者：宋巍 常青 陈皓 轩晨惠 赵旭芳 贾永森 SONG Wei;CHANG Qing;CHEN Hao;XUAN Chenhui;ZHAO Xufang;JIA Yongsen(Graduate School of North China University of Science and Technology,Tangshan 063210,Hebei,China;College of Traditional Chinese Medicine,North China University of Science and Technology,Tangshan 063210,Hebei,China)

机构地区：[1]华北理工大学研究生学院,河北唐山063210 [2]华北理工大学中医学院,河北唐山063210

出　　处：《现代中西医结合杂志》2020年第6期579-584,共6页Modern Journal of Integrated Traditional Chinese and Western Medicine

基　　金：河北省自然科学基金项目(H2017209053);河北省高等学校科学技术研究项目(QN2016013);河北省卫计委重点科技研究计划项目(20150493)

摘　　要：目的研究加味通幽汤对食管癌Eca-109细胞雷帕霉素靶蛋白(mTOR)/缺氧诱导因子1α(HIF-1α)通路及肿瘤缺氧相关因子的影响及干预机制。方法采用加味通幽汤、生理盐水分别灌胃大鼠14 d制备含药和对照血清。实验分为常氧组、缺氧组、常氧含药血清组和缺氧含药血清组,常氧组加入10%对照大鼠血清培养;缺氧组加入10%对照大鼠血清、10%CoCl 2缺氧诱导液培养;常氧含药血清组加入10%含药大鼠血清培养;缺氧含药血清组加入10%含药大鼠血清、10%CoCl 2缺氧诱导液培养。Western blot法检测各组中mTOR、HIF-1α及血管内皮生长因子(VEGF)、骨桥蛋白(OPN)、血管内皮细胞钙黏连蛋白(VE-cadherin)表达情况;实时荧光定量RT-PCR法检测各组中mTOR、HIF-1α、VEGF、OPN、VE-cadherin mRNA表达情况;免疫荧光法分析各组中VE-cadherin与HIF-1α共表达情况。结果Western blot分析显示,缺氧组细胞中mTOR、HIF-1α、VEGF、OPN、VE-cadherin蛋白表达水平均显著高于常氧组(P均<0.05),缺氧含药血清组mTOR、HIF-1α、VEGF、OPN、VE-cadherin蛋白表达水平均显著低于缺氧组(P均<0.05)。实时荧光定量RT-PCR检测显示,缺氧组细胞中mTOR、HIF-1α、VEGF、OPN、VE-cadherin mRNA相对表达水平显著高于常氧组(P均<0.05),缺氧含药血清组mTOR、HIF-1α、VEGF、OPN、VE-cadherin mRNA相对表达水平显著低于缺氧组(P均<0.05)。免疫荧光结果显示,缺氧组HIF-1α与VE-cadherin荧光强度较常氧组明显增强,缺氧含药血清组HIF-1α与VE-cadherin荧光强度较缺氧组均显著降低。结论加味通幽汤可显著抑制缺氧微环境下食管癌Eca109细胞mTOR/HIF-1α通路及肿瘤缺氧相关因子VEGF、OPN和VE-cadherin蛋白和mRNA表达。Objective It is to study the effect of modified Tongyou Decoction on mammalian target of rapamycin(mTOR)/hypoxia inducible factors 1α(HIF-1α)pathway and tumor hypoxia-related factors in esophageal cancer Eca-109 cells and its intervention mechanism.Methods The rats were respectively treated with modified Tongyou Decoction and normal saline by gavage for 14 d to prepare drug serum and control serum.The experiment was divided into normoxic group,hypoxia group,normoxic drug serum group and hypoxia drug serum group which were cultured with 10%control serum,10%control serum plus 10%CoCl2 hypoxia-induced liquid,10%drug serum and 10%drug serum plus 10%CoCl2 hypoxia-induced liquid.Western blot method was used to detect the expression of mTOR,HIF-1α,vascular endothelial growth factor(VEGF),osteopontin(OPN),and vascular endothelial cell cadherin(VE-cadherin)in each group;real-time fluorescence quantitative RT-PCR was used to detect the expressions of mRNA of mTOR,HIF-1α,VEGF,OPN,and VE-cadherin in each group;immunofluorescence was used to analyze the co-expression of VE-cadherin and HIF-1αin each group.Results Western blot analysis showed that the expression levels of mTOR,HIF-1α,VEGF,OPN,and VE-cadherin protein in the cells of hypoxic group were significantly higher than those of the normoxic group(P<0.05),and the expression levels of these indexes of hypoxia drug serum group were significantly lower than those of the hypoxia group(P<0.05).RT-PCR showed that the relative expression levels of mTOR,HIF-1α,VEGF,OPN,and VE-cadherin mRNA in the cells of the hypoxia group were significantly higher than those of the normoxic group(P<0.05),and the and the expression levels of these indexes of hypoxia drug serum group were significantly lower than those of the hypoxia group(P<0.05).The results of immunofluorescence showed that the fluorescence intensity of HIF-1αand VE-cadherin in the hypoxia group was significantly enhanced compared with the normoxic group,and the fluorescence intensity of HIF-1αand VE-cadherin in the hy

关 键 词：食管癌 加味通幽汤 缺氧 血管内皮生长因子 骨桥蛋白 血管内皮细胞钙黏连蛋白 

分 类 号：R-33[医药卫生]