金雀黄素对HEC-1B细胞增殖及荧光素酶报告基因表达影响的研究

摘要

Objective To investigate the influence and its receptor mechanism of genistein on the proliferation of negative cell line of estrogen receptor and progesterone receptor of endometrial carcinoma,HEC-1B and provide the theoretical base for the application of genistein in clinic. Methods HEC-lB were cultured in vitro, and than divided into the blank group, genistein group 1 (10×10-6 mol/L),genistein group 2 (20×10-6 mol/L), genistein group 3 (40×10-6 mol/L), genistein group 4 (80×10-6 mol/L) and genistein group 5 (160×10-6 mol/L) with different action time (24 hours, 48 hours,72 hours and 96 hours). The influence of genistein on the proliferation of HEC-1B was detected by using MTT method. The influences of genistein in different doses on the expressions of estrogen receptor-α (ER-α) and estrogen receptor-β(ER-β) regulated by estrogen response element (ERE) by applying the method of luciferase reporter gene. The difference between genistein effects on ER-α and ER-β were compared. Results The proliferation of HEC-1B in vitro was inhibited significantly by genistein with statistical difference (P ＜ 0.05). The inhibitory effect of genistein increased along with the increase of its concentration (10×10-6 mol/L - 160×10-6 mol/L) and action time (24 - 72 hours) showing a dose-time dependency. The expressions of reporter genes were much higher in the genistein group 2 and 3 than those in the blank group (P ＜ 0.05) showing a dose dependency. The increase of the expression of reporter gene mediated by ER-β was more significant that that by ER-α (P ＜ 0.01). Conclusion Genistein can inhibit the proliferation of endometrial carcinoma through regulating the expressions of ER-α and ER-β, and proportion of ER-β to ER-α.%目的 探讨金雀黄素对子宫内膜癌雌、孕激素受体阴性细胞系(HEC-lB细胞)增殖的影响及其受体作用机制,为临床运用金雀黄素抗肿瘤治疗提供理论依据.方法 体外培养子宫内膜癌HEC-lB细胞,金雀黄素作用浓度分别为10×10-6、20×l0-6、40× 10-6、80×l0-6、160×l0-6mol/L,作用时间分别为24、48、72、96 h.采用四甲基偶氮唑盐(MTT)法检测金雀黄素对子宫内膜癌细胞增值的影响;同时采用荧光素酶报告基因的方法,检测不同浓度金雀黄素对雌激素应答原件(ERE)调控的雌激素受体α(ER-α)和雌激素受体β(ER-β)报告基因表达的影响,并比较其对ER-α和ER-β作用的差异性.结果 金雀黄素对HEC-1B细胞体外增殖有明显抑制作用,差异有统计学意义(P＜0.01),其抑制作用随药物作用浓度增加和作用时间的延长而逐渐增强,呈剂量及时间依赖性.在金雀黄素浓度为20×10-6、40×10-6mol/L的作用下,报告基因的表达较空白对照组明显提高,差异有统计学意义(P＜0.01),且呈浓度依赖性;金雀黄素通过ER-β介导的报告基因表达水平的升高程度强于ER-α,差异有统计学意义(P＜0.01).结论 金雀黄素可抑制子宫内膜癌细胞的增殖,这种抑制作用可能通过调节子宫内膜癌细胞ER-α和ER-β的表达,调整ER-β/ER-α比例而实现.