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目的:观察雷帕霉素(Rapamycin)对人食管癌细胞TE1、TE13中HIF-1α的抑制作用及糖酵解途径相关基因表达的影响;分析mTOR信号通路与糖酵解途径的关系。方法:分别取TE1、TE13细胞株,常氧及缺氧条件下分别予以Rapamycin作用后,将两株细胞各分为四组:①正常对照组,即未处理组(N);②缺氧处理组(H);③常氧Rapamycin处理组(N+R);④缺氧Rapamycin处理组(H+R)(即雷帕霉素预处理1小时后缺氧培养)。采用实时定量PCR检测细胞中缺氧诱导因子-1α(Hypoxia-inducible factor-1α,HIF-1α)蛋白及己糖激酶Ⅱ(HK-Ⅱ)、葡萄糖载体蛋白1(GLUT-1)、乳酸脱氢酶A(LDHA)等糖酵解相关基因mRNA的表达,Western印迹检测HIF-1α及HK-Ⅱ、GLUT-1、LDHA等糖酵解相关基因蛋白的表达。结果:缺氧处理后与正常对照组相比,HIF-1α、HK-Ⅱ、GLUT-1蛋白表达及mRNA明显增强,而LDHA表达无明显变化;Rapamycin预处理后,正常对照组及缺氧处理组HIF-1α、HK-Ⅱ、GLUT-1的表达均明显降低,LDHA无明显变化。结论:常氧及缺氧条件下,雷帕霉素可抑制食管癌细胞HIF-1α及糖酵解相关基因的表达,提示阻断mTOR通路可抑制食管肿瘤细胞的糖酵解途径。
Objective: To observe the inhibitory effect of rapamycin on human esophageal cancer cells TE1, TE13 HIF-1α and glycolytic pathway related gene expression; analysis mTOR signaling pathway and glycolysis pathway. Methods: The TE1 and TE13 cell lines were respectively treated with Rapamycin under normoxia and anaerobic conditions. The two cell lines were divided into four groups: ① normal control group, untreated group (N); ② hypoxic treatment (H); ③ normoxia Rapamycin treatment group (N + R); ④ hypoxia Rapamycin treatment group (H + R) (ie rapamycin pretreatment after 1 hour hypoxia culture). The expression of Hypoxia-inducible factor-1alpha (HIF-1alpha), hexokinase II (HK-II), glucose transporter 1 (GLUT- LDH and other glycolytic related gene mRNA expression, Western blot detection of HIF-1αand HK-Ⅱ, GLUT-1, LDHA and other glycolytic related gene protein expression. Results: Compared with the normal control group, the expression of HIF-1α, HK-Ⅱ, GLUT-1 protein and mRNA were significantly increased after hypoxia treatment, while the expression of LDHA did not change significantly. After Rapamycin preconditioning, The expression of HIF-1α, HK-Ⅱ, GLUT-1 in the group was significantly lower than that in the control group. Conclusion: Under normoxia and anaerobic conditions, rapamycin can inhibit the expression of HIF-1α and glycolysis-related genes in esophageal cancer cells, suggesting that blocking the mTOR pathway may inhibit the glycolytic pathway of esophageal cancer cells.