中文摘要：

目的 探讨雌二醇诱导子宫内膜癌细胞系Ishikawa细胞生成的血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）对有丝分裂原活化蛋白激酶（MAPK）通路中主要基因蛋白的影响.方法 实验分为4组：雌二醇组（E2组,加入1 μmol/L的雌二醇作用30 min）,抑制剂组[包括Bibf1120组：加入10 μmol/L的VEGF受体抑制剂——Bibf1120; Ponatinib组：加入2.5 μmol/L的bFGF受体抑制剂——Ponatinib; U0126组：加入10μmol/L的MAPK激酶（MEK）特异性抑制剂——U0126.各组均预处理1 h],抑制剂＋E2组（包括Bibf1120＋E2组、Ponatinib＋E2组和U0126＋E2组,各抑制剂预处理1h后,加入1μmol/L的雌二醇作用30 min）;对照组（仅加入无血清DMEM培养基）.（1）以不同浓度（分别为0.01、0.1、1、10、100μmol/L）雌二醇作用于Ishikawa细胞后,采用蛋白印迹法检测磷酸化细胞外信号调节激酶1/2（p-ERK1/2）蛋白活化水平.（2）采用荧光定量PCR技术检测各组细胞中VEGF、bFGF、MEK1/2、ERK1/2mRNA的表达,蛋白印迹法检测各组细胞中VEGF、bFGF蛋白的表达及磷酸化NEK1/2（p-MEK1/2）、p-ERK1/2蛋白活化水平,流式细胞仪检测各组细胞的细胞周期比例,体外穿膜实验检测各组细胞的迁移能力.结果 （1）不同浓度（分别为0.01、0.1、1、10、100 μmo1/L）的雌二醇作用30 min后Ishikawa细胞中p-ERK1/2蛋白的活化水平分别为0.16±0.03、0.10±0.03、0.41±0.04、0.19±.0.03、0.19±0.03,均明显高于对照组的0.05±0.00 （P＜0.05）,且雌二醇浓度为1μmol/L时其活化水平最高.（2）E2组细胞中VEGF、bFGF mRNA及蛋白的表达水平均高于对照组,分别与对照组比较,差异均有统计学意义（P＜0.05）;Bibf1120＋E2组细胞中VEGF mRNA及蛋白的表达水平分别与E2组比较,差异均有统计学意义（P＜0.05）.E2组MEK1/2、ERK1/2mRNA及p-MEK1/2、p-ERK1/2蛋白活化水平均显著高于对照组（P＜0.05）;Bibf1120＋E2组、Ponatinib＋E2?

英文摘要：

Objective To explore the effects of mitogen-activated protein kinase （MAPK） pathway by estradiol induced vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF） in endometrial cancer Ishikawa cells.Methods The experiments were divided into 4 groups：E2 group （Ishikawa cells treated with 1 p mol/L estradiol for 30 minutes）; inhibitor group：including Ishikawa cells treated with 10 μmol/L Bibf1 120 （Bibf1 120 group）,or treated with 2.5 μmol/L Ponatinib （Ponatinib group）,or treated with 10 p mol/L U0126 （U0126 group） for 60 minutes; inhibitor ＋ E2 group：including Ishikawa cells treated with 10 μmol/L Bibf1120 （Bibf1120 ＋ E2 group）,or treated with 2.5 μmol/L Ponatinib （Ponatinib ＋ E2 group）,or treated with 10 μmol/L U0126 （U0126＋ E2 group） for 60 minutes following incubation with 1 μmol/L estradiol for 30 minutes; control group：only adding the culture medium without serum DMEM.（1） Western blot analysis was used to detect phosphorylation extracellular signal-regulated kinase 1/2（p-ERK 1/2） protein expression with stimulation in different concentrations of estradiol （0.01,0.1,1,10,100 μmol/L）.（2） Quantitative fluorescent reverse transcription （qRT）-PCR and western blot analysis was used to test the level of mRNA and protein of VEGF,bFGF,MAPK kinase 1/2 （MEK1/2）,extracellular signal-regulated kinase 1/2 （ERK1/2）,p-ERK1/2 and phosphorylation MEK1/2 （p-MEK1/2）.Flow cytometry were used to examine the cell cycle,and transwell chamber assay were used to detect the cell migration in different groups.Results The expression of the p-ERK1/2 protein at 0.01,0.1,1,10,100 μ mol/L were 0.16±0.03,0.10±0.03,0.41 ±0.04,0.19±0.03,0.19±0.03,there were significantly higher than that in control group（0.05±0.00,P＜0.05）,and which was more obvious at the concentration of 1 μmol/L estradiol.The expression level of VEGF,bFGF mRNA and protein in E2 group were higher than those in the control group （P＜O.05）.