【作者】 秦定霞；

【导师】 刘嘉茵；

【作者基本信息】 南京医科大学 ， 妇产科学， 2010， 硕士

【摘要】 背景和研究目的:双酚A (Bisphenol A,BPA)是塑料工业生产聚碳酸酯、环氧树脂、酚醛树脂等物质的前体物质,广泛应用于染料及医疗器械、食品包装材料、婴儿奶瓶及牙科填充剂等塑料工业。研究发现,BPA是一种环境内分泌干扰物,流行病学调查显示成年男性及女性、孕妇及儿童血中游离BPA的浓度在0.3～4.4ng/ml;据报道,BPA浓度在多囊卵巢综合征(PCOS)、习惯性流产患者血清中明显升高。动物实验表明,BPA可通过与雌激素受体(ER)结合等多种信号通路影响雌性生殖系统的形态及功能。但是,BPA对人类生殖系统的影响及相关机制至今尚未完全阐明。本研究通过体外培养人子宫内膜基质细胞(hESCs)及子宫内膜腺癌细胞株(RL95-2),观察BPA对子宫内膜细胞周期的影响,探讨BPA对子宫内膜细胞作用的机制,为进一步阐明BPA在生殖系统疾病的发生发展中的作用提供理论依据。方法:(1)参照实验室前期方法,分离获取hESCs,用细胞免疫荧光的方法对hESCs进行形态及纯度鉴定:波形蛋白特异性地标记hESCs,角蛋白特异性标记腺上皮细胞,体外培养和传代。(2)无血清培养24小时后,给予不同浓度的BPA(10-8, 10-6, 10-4mol/L)处理hESCs 24h,以10-8 mol/L的E2为阳性参照。(3)流式细胞技术测定不同浓度BPA处理组hESCs细胞周期的改变。(4) Trizol法裂解细胞提取总RNA,Real-time PCR法测定不同浓度BPA处理后hESCs ERα和AR mRNA表达水平。(5)进一步利用RL95-2细胞验证BPA对人子宫内膜细胞的影响:细胞免疫荧光的方法验证RL95-2细胞株ERα的表达;不同浓度BPA处理RL95-2细胞24h,流式细胞技术测定RL95-2细胞周期的改变。Real-time PCR法测定RL95-2细胞ERαmRNA表达水平;免疫印迹法测定ERα蛋白表达量;(6)通过检测不同浓度BPA处理条件下RL95-2细胞ER相关信号通路的关键因子的表达变化,深入探讨BPA影响子宫内膜细胞可能的作用机制:不同浓度的BPA (10-8, 10-6, 10-4mol/L)处理RL95-2细胞20min后,免疫印迹法对RL95-2细胞Akt/P-Akt, ERK1/2/P-ERK1/2, p38/ P-p38蛋白水平的表达进行研究,并在BPA处理的同时加用ER阻断剂ICI182 780, ERK信号通路阻断剂PD98059预处理90min,检测相关因子的表达。结果:(1)细胞免疫荧光:角蛋白在基质细胞染色中呈阴性,波形蛋白染色阳性率达95%。(2)流式细胞技术检测不同浓度BPA处理24h后hESCs细胞周期,结果显示与阴性对照组相比,10-4mol/L BPA处理组hESCs生长阻滞于G1期(P<0.05),S期及G2期均无影响。(3)不同浓度BPA处理hESCs 24h后,与阴性对照组相比,ERαmRNA表达水平在各浓度BPA(10-8, 10-6, 10-4mol/L)处理组均明显下降(P<0.05),而在E2组表达明显升高(P<0.05)。与阴性对照组相比,AR mRNA表达水平在10-4mol/L BPA处理组明显升高(P<0.05),在10-6, 10-8mol/L BPA处理组有增高趋势(P>0.05)。(4)不同浓度BPA处理RL95-2细胞24h后,与阴性对照组相比,10-6, 10-4mol/LBPA处理组ERαmRNA表达水平明显下降(P<0.05),10-8mol/L BPA处理组ERαmRNA表达有下降趋势(P>0.05),E2组ERαmRNA表达明显升高(P<0.05);免疫印迹结果显示,与阴性对照组相比,10-6, 10-4mol/L BPA处理组ERα蛋白表达量明显下降(P<0.05),10-8mol/L BPA处理组ERα蛋白表达量无明显差异;E2组ERα蛋白表达量明显升高(P<0.05)。(5) ICI182 780预处理90min,BPA作用RL95-2细胞24h后,和单用10-6 mol/L BPA组相比,10-6 mol/LBPA+ ICI182 780组ERα蛋白表达量无明显差异,ICI182 780可抑制E2引起的ERα蛋白表达量的增加。(6)不同浓度BPA处理RL95-2细胞20min后:各浓度BPA处理组均能够明显增加RL95-2细胞ERK1/2的磷酸化水平(P<0.05),当用PD98059预处理90min后,BPA引起的ERK1/2的磷酸化被明显抑制;不同浓度的BPA处理RL95-2细胞20min后,p38, Akt蛋白磷酸化水平均无明显改变。结论:(1)一定剂量的BPA可将子宫内膜细胞周期阻滞在G1期,影响细胞的存活与生长。BPA可引起人子宫内膜细胞甾体激素受体基因及蛋白水平的改变,且这种改变具有浓度依赖性。(2) BPA可增加ERK1/2蛋白磷酸化,通过“非基因组效应”(膜雌激素受体),影响人子宫内膜细胞的存活与生长,并引起甾体激素受体基因及蛋白水平的改变。(3)本研究为揭示环境内分泌干扰物BPA对人类子宫内膜结构与功能的影响提供了进一步的证据。更多还原

【Abstract】 Objective Bisphenol A (BPA), a monomer of polycarbonate plastics, is one of the highest volume chemicals used in industry. Polycarbonates, including BPA, are used in numerous consumer products, including food and water containers, baby bottles, linings of metal food and beverage cans, medical tubing, epoxy resins, and dental fillings. BPA is an endocrine disrupting chemicals. Epidemiological survey show, levels of BPA ranging from 0.3 to 4.4 ng/ml are present in adult men and women, pregnant women and children,. Serum BPA concentrations were significantly higher in women with polycystic ovary syndrome (PCOS) and spontaneous abortion. Several studies have documented that BPA may act by binding to estrogen receptor and then affect the morphology and function of female reproductive system. However, the mechanism of BPA on human reproductive system has not yet been clarified. In this study, human endometrial stroma cells (hESCs) and RL95-2 cells were cultured in vitro, the effect of BPA on proliferation of human endometrial cells and both estrogen receptor (ERα) and androgen receptor (AR) mRNA expression were observed. Adding strong support to the role and mechanism of BPA on female reproductive system.Methods: (1) The hESCs were isolated, Reference laboratory method previously. Using immunofluorescence methods to identify the hESCs formation and purity: Vimentin specifically labeled stromal cells, Keratin was epithelial cells specific marker, then cultured and passaged,(2) Cultured cells were treated with gradient concentrations of BPA (10-8, 10-6, 10-4mol/L) after synchronization by using serum-free culture medium for twenty-four hours. (3) Flow Cytometry were used to detect the cell cycles. (4) Total RNA of the cultured hESCs were extracted by Trizol and the mRNA expression of ERαand AR were detected by real-time PCR. (5) Using endometrial adenocarcinoma cell line (RL95-2) to explore the possible mechanism of BPA on human endometrial: Using immunofluorescence methods to identify the expression of ERαon RL95-2; Flow Cytometry was used to detect the cell cycles; ERαmRNA was detected by real-time PCR; ERαprotein were detected by western blot. (6) Twenty minutes after treatment with BPA, the protein expression of Akt/P-Akt, ERK1/2/P-ERK1/2, p38/P-p38 were detected by western blot, at the same time plus ERαantagonist ICI182 780 or ERK pathway inhibitor PD98059.Result: (1) Immunochistochemical staining: Keratin staining in stromal cells were negative, Vimentin positive staining rate reach 95%.(2) Twenty-four hours after treatment with BPA, compared with control, the hESCs cell cycles were significantly inhibited by BPA at the concentration of 10-4 mol/L (P<0.05). (3) Twenty-four hours after treatment with BPA, ERαmRNA expression in cultured hESCs was detected by real-time PCR, compared with control, ERαmRNA expression was down-regulated with gradient concentrations of BPA (P<0.05), ERαmRNA expression was significantly higher at E2 group (P<0.05), compared with control, AR mRNA was significantly increased with BPA at the concentration of 10-4mol/L, AR mRNA were observed to have the trend to be increased when the concentration of BPA were 10-6, 10-8mol/L, although there were no statistical significance (P>0.05). (4) Twenty-four hours after the RL95-2 cells treatment with BPA, compared with the control, ERαmRNA expression was down-regulated with 10-6, 10-4 mol/L of BPA (P<0.05), ERαmRNA expression was observed to have the trend to be down-regulated when the concentration of BPA was 10-8mol/L, ERαmRNA expression was significantly higher at E2 group (P<0.05); ERαprotein was detected by western blot, compared with control, ERαprotein was down-regulated with 10-6, 10-4 mol/L of BPA (P<0.05), ERαprotein was significantly higher at E2 group (P<0.05). (5) After treating with 10-6 mol / L BPA + ICI182 780 group and 10-6 mol / L BPA, ERαprotein in RL95-2 cells was no significant difference, however, ERαprotein in cultured RL95-2 cells was down-regulated with E2 + ICI182 780. (6) Twenty minutes after gradient concentrations of BPA, the activation of ERK1/2 phosphorylation was significantly higher (P<0.05), the phosphorylation levels of ERK1/2 was significantly inhibited with PD98059 + BPA; on the other hand, the phosphorylation level of Akt, p38 protein did not change significantly.Conclusion:(1) After the treatment with BPA, hESCs and RL95-2 cell cycle were arrested at G1 phase, affecting cells survival and growth. (2) BPA can change human endometrial cells steroid receptors gene and protein level, and this alter have concentration-dependent. (3) After treating with BPA, the activation of ERK1/2 phosphorylation was significantly higher, affecting cells survival and growth, altered human endometrial cells structure and function through“non-genomic effect”.更多还原