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目的:探讨细胞外调节蛋白激酶(ERK)在雌激素促乳腺癌细胞MCF-7增殖、细胞周期转化中的作用及机制。方法:以雌激素受体阳性乳腺癌细胞MCF-7为研究对象,MTT法检测17β-雌二醇(17β-E2)对MCF细胞增殖的影响,确定实验处理用浓度;MTT法检测PD98059对17β-E2促MCF-7细胞增殖作用的影响,求取其中效抑制浓度;流式细胞术检测细胞周期;TRAP-PCR银染法检测端粒酶活性,Western印迹法检测p-ERK1/2、野生型p53蛋白水平;RT-PCR检测野生型p53 mR-NA水平。结果:ERK磷酸化抑制剂PD98059能抑制17β-E2对MCF-7细胞的促增殖作用,具有时效-量效关系,其差异具有显著统计学意义(P<0.01),各作用时间点PD98059中效抑制浓度分别为89.28μmol/L.24 h、39.81μmol/L.48 h、21.87μmol/L.72 h。应用PD98059 48 h后,能抑制17β-E2促进MCF-7细胞周期转化作用,使G1期细胞增加,S期和G2期细胞减少(P<0.01);阻抑17β-E2增强MCF-7细胞端粒酶活性的作用(P<0.05);增强野生型p53蛋白表达和p53基因转录水平(P<0.01);p-ERK1/2蛋白表达水平显著降低(P<0.01)。结论:ERK在17β-E2促乳腺癌细胞MCF-7增殖、细胞周期转化中具有重要的作用,其机制与野生型p53转录和端粒酶活性改变有关。
AIM: To investigate the role and mechanism of extracellular regulated protein kinase (ERK) in the proliferation and cell cycle of estrogen-induced breast cancer MCF-7 cells. Methods: The estrogen receptor positive breast cancer cell line MCF-7 was used as the research object. The effect of 17β-estradiol (17β-E2) on the proliferation of MCF cells was determined by MTT assay. The concentration of experimental treatment was determined. The effect of PD98059 on 17β -E2 to promote the proliferation of MCF-7 cells, and the inhibitory concentrations of p-ERK1 / 2 were determined by flow cytometry; the telomerase activity was detected by TRAP-PCR silver staining; p-ERK1 / P53 protein level; RT-PCR detection of wild-type p53 mR-NA levels. Results: ERK phosphorylation inhibitor PD98059 could inhibit the effect of 17β-E2 on proliferation of MCF-7 cells, and had a time-dose-effect relationship. The difference was statistically significant (P <0.01), PD98059 at various time points The inhibitory concentrations were 89.28μmol / L for 24 h, 39.81μmol / L for 48 h and 21.87 μmol / L for 72 h, respectively. After PD98059 was administered for 48 h, 17β-E2 could inhibit the cell cycle transformation of MCF-7 cells, increase the cells in G1 phase and decrease the cells in S phase and G2 phase (P <0.01) (P <0.05); enhance the expression of wild-type p53 protein and p53 gene (P <0.01); and the expression of p-ERK1 / 2 protein decreased significantly (P <0.01). Conclusion: ERK plays an important role in the proliferation and cell cycle of 17β-E2-induced breast cancer cell line MCF-7. The mechanism is related to the change of wild-type p53 and telomerase activity.