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目的研究表皮生长因子 (EGF)在子宫内膜癌细胞信号转导中 ,激活细胞外信号调节激酶 (ERK)的情况 ,探讨抑癌蛋白PTEN在EGF诱导的对ERK的活化作用。方法构建野生型PTEN和突变型PTEN(G12 9E)编码基因的逆转录病毒载体 ,体外转染子宫内膜癌细胞Ishikawa ,用G4 18筛选稳定表达的细胞系。用Westernblot检测PTEN基因在Ishikawa细胞中的表达 ,以及EGF诱导Ishikawa EGFP、Ishikawa PTEN和Ishikawa PTEN(G12 9E)中活化ERK的浓度效应和时相特点。结果EGF可激活ERK ,但以激活ERK2为主。以不同浓度的EGF作用于Ishikawa EGFP和Ishikawa PTEN时 ,10 μg/L的EGF可使ERK达到最大活化 ,刺激 5min活化最明显。与Ishikawa EGFP相比较 ,EGF对Ishikawa PTEN中ERK的活化作用明显减弱 ,但未降低到阴性对照的水平。 10 μg/L的EGF刺激Ishikawa PTEN(G12 9E) 5min ,与Ishikawa EGFP相比较无明显改变。结论EGF可刺激子宫内膜癌细胞ERK活化 ,而脂质磷酸酶PTEN则能抑制EGF诱导的ERK活化
Objective To investigate the activation of extracellular signal-regulated kinase (ERK) by epidermal growth factor (EGF) in endometrial cancer cell signal transduction and to explore the role of tumor suppressor protein PTEN in EGF-induced activation of ERK. Methods Retroviral vectors encoding wild-type PTEN and mutant PTEN (G129E) were constructed and transfected into Ishikawa endometrial carcinoma cells in vitro. Stably expressing cell lines were screened by G418. The expression of PTEN gene in Ishikawa cells was detected by Western blot and the concentration-effect and time-phase characteristics of EGF-induced activation of ERK in Ishikawa EGFP, Ishikawa PTEN and Ishikawa PTEN (G129E). Results EGF can activate ERK but mainly activate ERK2. With different concentrations of EGF on Ishikawa EGFP and Ishikawa PTEN, 10 μg / L EGF can make ERK reach the maximum activation, stimulated 5min activation most obvious. The activation of ERK in Ishikawa PTEN was significantly attenuated by EGF compared to Ishikawa EGFP, but not down to the negative control. 10 μg / L EGF stimulated Ishikawa PTEN (G12 9E) for 5 min, no significant change compared with Ishikawa EGFP. Conclusion EGF can stimulate the activation of ERK in endometrial cancer cells, whereas PTEN, a lipid phosphatase, can inhibit the activation of ERK induced by EGF