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题名: 全氟碘烷调控ERα/β 介导的乳腺癌细胞增殖效应研究
作者: 宋文婷1
学位类别: 博士后
答辩日期: 2017-05
授予单位: 中国科学院大学
授予地点: 北京
导师: 江桂斌 ; 周群芳
关键词: 内分泌干扰物 ; 全氟碘烷 ; Endocrine disrupting chemicals ; ER/ ; Polyfluorinated iodine alkanes ; 乳腺癌细胞 ; ER/ ; 增殖 ; breastcancer cells ; proliferation
学位专业: 环境科学
中文摘要: 全氟碘烷是一类新型内分泌干扰物,基于酶片断互补技术,本文建立了一种适用 于内分泌干扰物筛选的ER竞争结合方法,利用此方法研究了包括全氟己基碘烷 (PFHxI)、全氟辛基碘烷(PFOI)、1,6-二碘全氟己烷(PFHxDI)和1,8-二碘代全 氟辛基碘烷(PFODI)在内的四种全氟碘烷与ERα和ERβ的结合能力。该方法可稳定 化学发光9 h,在ERα和ERβ浓度为40 nM时化学发光信号对ER竞争响应最灵敏。通过 阳性对照E2和染料木黄酮的研究结果可以表明,该方法可有效识别化合物与ERα和 ERβ的结合能力差异。基于该方法对四种全氟碘烷的评价表明,它们对ERα和ERβ表 现出不同的结合能力与结合倾向。全氟碘烷与两种ER亚型的结合能力顺序为PFHxDI > PFODI > PFHxI > PFOI。PFOI、PFHxDI和PFODI与ERα的结合能力更强,而PFHxI与 ERβ的结合能力更强。PFOI、PFHxDI和PFODI竞争结合ERα曲线与MVLN检测结果一 致。PFHxI没有引起MVLN细胞荧光素酶响应的原因可能为PFHxI与ERα结合能力较弱 或降低了MVLN细胞转录活性。该受体竞争结合方法具有稳定、简单、快速、高通量 等优点,且可以识别化合物与ERα和ERβ的相对结合能力,可适用于大规模筛选具有 类雌激素活性的内分泌干扰物,并阐释其通过不同ER亚型调控的毒性作用机制,对 揭示化合物潜在的毒性效应和暴露风险具有重要意义。 根据四种全氟碘烷与ERα和ERβ相对结合能力的不同,选择优势结合ERα的 PFHxDI和优势结合ERβ的PFHxI作为代表性化合物,采用细胞活力检测、EdU探针分 析细胞增殖和细胞周期检测实验,研究了它们对两种ERα/ERβ比例不同的ER阳性乳 腺癌细胞MCF-7和T47D的增殖效应。结果表明100 μM和200 μM PFHxDI对MCF-7和 T47D细胞表现出明显的毒性效应,200 μM PFHxI显著降低了ERα/ERβ比例高的 MCF-7的细胞活力,而没有显著影响ERα/ERβ比例低的T47D的细胞活力。由此可见, 不同ER选择性配体对ERα/ERβ表达比例不同的乳腺癌细胞毒性效应可能不同,即ER 表达较高的MCF-7对高剂量的ER选择性配体的暴露较为敏感。基于EdU探针分析显 示,E2(0.05 nM)、PFHxI(5 μM)、PFHxDI (0.02 ,0.2 μM)暴露可诱导MCF-7 细胞增殖能力显著升高,相对而言,仅有E2(0.05 nM)与PFHxDI(0.2 μM)暴露显 著增加了T47D细胞的增殖能力,且雌激素化合物暴露对MCF-7增殖能力的提高程度 大于T47D,表明MCF-7对雌激素化合物暴露更为敏感,这可能与ERα可调控乳腺癌细 胞的增殖作用有关。作为与ERβ有着更强结合力的选择性配体,PFHxI(50 μM)可诱导T47D的增殖,但对MCF-7的生长表现出一定的抑制效应,MCF-7细胞DNA合成 抑制可能是由于该化合物调控MCF-7中ERβ介导的信号转导从而抑制了ERα介导的细 胞增殖效应。两种暴露细胞的细胞周期分析结果显示,除50 μM PFHxI暴露导致MCF-7 细胞(S+G2/M)期比例降低外,其它暴露组均诱导该类细胞S期比例显著上升,表明 雌激素类化合物对ER表达较高的乳腺癌细胞的增殖效应。对于T47D细胞,PFHxI 主要增加其G2/M期细胞诱导细胞增殖现象,而PFHxDI主要是通过增加S期细胞比例 引起细胞增殖。PFHxI和PFHxDI导致T47D细胞增殖机制的不同可能归因于其对细胞 中ERα和ERβ的选择性结合程度不同所致。细胞周期实验结果很好印证了EdU探针分 析数据。总之,雌激素化合物对细胞的增殖效应依赖于细胞中ERα/β的表达水平和雌 激素化合物与ERα和ERβ结合能力的强弱。该研究表明可疑环境因子对不同类型乳腺 癌发生发展调控过程具有潜在的影响。 总之,本博后工作围绕新型环境污染物内分泌干扰效应筛选,建立了高通量、灵 敏有效的能识别不同ER亚型结合能力的类雌激素污染物鉴别方法。结合ERα/ERβ表 达比例不同的乳腺癌增殖效应研究,评价了两种结合ERα或ERβ能力不同的代表性全 氟碘烷暴露引起的细胞增殖效应,研究发现可望为不同类型乳腺癌发生发展过程的调 控提供科学思路
英文摘要: Polyfluorinated iodine alkanes (PFIs) are emerging endocrine disrupting chemicals (EDCs). A novel competitive binding assay based on enzyme fragmentation complementation technology was established to screen the binding affinities of emerging EDCs for estrogen receptor (ER) α or β isoforms. Binding affinities of four PFIs, including tridecafluorohexyl iodide (PFHxI), heptadecafluoro-n-octyl iodide (PFOI), dodecafluoro-1,6-diiodohexane (PFHxDI) and hexadecafluoro-1,8-diiodooctane (PFODI), for ERα and β were screened by the competitive binding assay. It was found that luminescent signals were sustainably emitted for 9 h, and 40 nM ERα or β in the system would lead to the most sensitive luminescence response. Using 17β-estrodiol (E2) and genistein as the representative estrogenic hormones, their binding affinities for ERα and β were evaluated. The results were consistent with those determined by traditional methods. Four PFIs showed diverse binding affinities and different preferences to ERα or β isoforms. The order of binding affinities of four PFIs for ER was PFHxDI > PFODI > PFHxI > PFOI. PFOI, PFHxDI and PFODI showed higher binding affinities for ERα and PFHxI showed higher binding affinity for ERβ. The binding affinities of PFOI, PFHxDI and PFODI for ERα were consistent with the result from MVLN transcriptional reporter assay. PFHxI failed to induce obvious luciferase response in MVLN cells due to its low binding affinity for ERα or decreased transcriptional activity in the cells. The newly developed competitive binding assay presented in this study was stable, simple and rapid and could evaluate binding affinities of emerging chemicals for ER isoforms. The competitive binding assay provided a promising alternative to high throughput screening of emerging chemicals with estrogenic effects and revealing useful information on their ER binding mechanism, which would be of great importance in explanation of their potential toxicological effects and human exposure risks. Based on the different binding preferences of PFIs, PFHxDI and PFHxI were selected as two representative estrogenic EDCs preferentially binding to ERα and ERβ, respectively. The effects of PFHxI and PFHxDI on two kinds of breast cancer cells with different ERα/β expression levels, MCF-7 and T47D, were evaluated by cell viability, proliferation analysis using EdU probe, and cell cycle assay. The results showed that 100 μM and 200 μM PFHxDI significantly decreased cell viabilities in both MCF-7 and T47D, whereas 200 μM PFHxI only resulted in significant decrease in the cell viability of MCF-7, and showed no obvious influences on T47D cells. It suggested that ligands with different binding selectivities for ERα or β could exhibit different cytotoxicities to MCF-7 and T47D. MCF-7 with high ERα expression levels in cells was more sensitive to high dose of the ligand preferentially binding to ERβ. Based on EdU probe analysis, the treatments of E2 (0.05 nM), PFHxI (5 μM), or PFHxDI (0.02, 0.2 μM) significantly increased the proliferation of MCF-7, while T47D proliferation was significantly induced only by the stimulations of E2 (0.05 nM) or PFHxDI (0.2 μM). Apparently, MCF-7 was more volunerable to estrogenic compounds exposure when compared with T47D regarding cell proliferation. This was attributed to the relatively higher ERα expression in MCF-7, which directly regulated cellular proliferation. PFHxI (50 μM) induced T47D proliferation, but inhibited MCF-7 proliferation, which could be related with ER-mediated inhibition in cellular DNA synthesis regulated by ERα. Based on flow cytometry, the percentage of S+G2/M cells was decreased in 50 M PFHxI-exposed MCF-7. The cell numbers in S phase were significantly increased in the other MCF-7 exposure groups with the stimulations of PFHxI and PFHxDI, showing the enhanced cell proliferation. Comparatively, in exposure system of T47D, PFHxI induced increased G2/M phase cells, while PFHxDI elevated S phase cells. This difference could be caused by different binding preferences of these two chemicals for ERα and ERβ in T47D. The results from flow cytometry were well consistent with those observed in EdU probe assay. Overall, the effects of estrogen-like compounds on cell proliferation were dependent on ERα/ERβ expression levels in cells and potentials of chemicals in binding to ER isoforms. The findings obtained herein provided evidences on the potentials of some suspicious environmental factors in regulation of the development and progression of different breast cancers. In conclusion, a sensitive, effective and high throughput screening assay was establis to screen the binding affinities of emerging EDCs for estrogen receptor (ER) α or β isoforms in this study. Using PFHxDI and PFHxI as the representative ligands preferentially binding to ERα and ERβ, their stimulative effects on proliferations of two breast cancer cells with different ERα/ERβ expressions were evaluated. The findings obtained in this study gave useful information on emerging EDC-induced effects in the development and progression of different breast cancers.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38690
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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作者单位: 1.中国科学院生态环境研究中心

Recommended Citation:
宋文婷. 全氟碘烷调控ERα/β 介导的乳腺癌细胞增殖效应研究[D]. 北京. 中国科学院大学. 2017.
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