目的 研究子宫内膜癌Ishikawa细胞中17β雌二醇通过细胞膜受体对丝裂原活化蛋白激酶(MAPK)通路的快速激活效应.方法 17β雌二醇偶联牛血清白蛋白(E2-BSA)在不同的时间作用于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(ERK)1和ERK2(ERK1/2)的表达情况,并利用激光扫描共焦显微镜(激光共聚焦)技术观察雌激素是否可以与细胞膜受体结合,不穿过细胞膜而快速磷酸化ERK1/2.结果 蛋白印迹法检测发现,ERK1/2在1×10-7mol/L的E2-BSA作用5 min后被快速磷酸化,磷酸化ERK1/2蛋白的表达水平为0.52,60 min时达到高峰(表达水平为0.98),120 min时回落(表达水平为0.09).激光共聚焦技术显示,E2-BSA在作用5 min时与细胞膜上相应受体结合,未穿过细胞膜即增高磷酸化ERK1/2的表达.结论 雌激素可以通过细胞膜受体快速激活子宫内膜癌细胞的MAPK通路.%Objective To study the nongenomic effect on mitogen-activated kinase (MAPK) signal transduction of 17β-estradiol at plasma membrane in endometrial cancer cell Ishikawa. Methods Estradiol-bovine serum albumin conjugates (E2-BSA) was added to Ishikawa cells at different time points. Western blot was used to detect the protein expression of phosphorylated extracellular signal-regulated kinase (ERK) 1/2. Confocal was used to verify if the phosphorylation of ERK1/2 was initiated at the membrane. Results Phosphorylation of ERK1/2 was elicited after 5 minute exposure to 1×10-7 mol/L E2-BSA. It was induced to a maximum following 60 minute exposure and descended after 2 hour. The expression levels of phospborylated ERK1/2 were 0. 52, 0. 98, and 0.09, respectively. Confocal proved that E2-BSA combined with membrane receptors after 5 minutes and elevated the phosphorylation of ERK1/2. Conclusion Membrane actions of estrogen can activate the rapid signaling cascades of MAPK in endometrial carcinoma cells.
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