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目的:研究GPER(G protein-coupled estrogen receptor,GPER)抑制剂对雌激素(estrogen,E2)长期作用下乳腺上皮细胞生长和克隆形成的影响,探讨阻断GPER受体防治乳腺癌的可能性。方法:分别使用E2、E2+G15和G15持续作用MCF-12A细胞5周(共传11代)建立细胞模型,通过光镜观察细胞形态的变化、台盼蓝计数法分析细胞生长变化,采用Western blot检测雌激素受体α(estrogen receptorα,ERα)及GPER蛋白表达变化,软琼脂糖克隆形成实验分析细胞克隆形成能力。结果:(1)MCF-12A经E2(10,20和40 nmol·L~(-1))连续处理5周后,其体外生长能力与E2浓度呈一定剂量依赖性,其中,E2(20 nmol·L~(-1))组细胞呈多层重叠样生长,排列紊乱,形态变化最为显著。(2)相比于对照组,E2组中不同代数细胞的ERα蛋白表达明显下调,且呈一定时间依赖性,而GPER蛋白表达未见明显变化。(3)GPER抑制剂G15能抑制E2诱导的细胞生长。(4)G15抑制E2诱导的细胞克隆形成。结论:G15可抑制E2对乳腺上皮细胞MCF-12A的促生长及克隆形成作用,提示GPER抑制剂可能可作为乳腺癌的防治药物。
OBJECTIVE: To study the effect of GPER inhibitors on the growth and colony formation of mammary epithelial cells under the action of estrogen (E2) for a long period of time and to explore the possibility of blocking the GPER receptor in the prevention and treatment of breast cancer. Methods: The cell model was established by continuous administration of E2, E2 + G15 and G15 for 5 weeks (11 passages in total), the changes of cell morphology were observed by light microscope, the cell growth was analyzed by trypan blue counting, The expression of estrogen receptorα (ERα) and GPER protein was detected by Western blotting. The colony formation ability of the cells was analyzed by soft agarose colony forming assay. Results: (1) The growth of MCF-12A in vitro was dose-dependent with the concentration of E2 after continuous treatment with E2 (10, 20 and 40 nmol·L -1) for 5 weeks. Among them, E2 · L ~ (-1)) cells showed overlapping growth in multiple layers, with disorder and the most obvious morphological changes. (2) Compared with the control group, the expression of ERα protein in different algebraic cells in E2 group was significantly down-regulated, and the expression of GPER protein showed no significant change. (3) GPER inhibitor G15 can inhibit E2-induced cell growth. (4) G15 inhibits E2-induced cell clone formation. Conclusion: G15 can inhibit the growth of mammary epithelial cells MCF-12A and the formation of colony formation, suggesting GPER inhibitors may be used as a preventive drug for breast cancer.