本文關(guān)鍵詞： 低劑量 鄰苯二甲酸二乙基己酯 人乳腺癌細(xì)胞MCF-7 PGE_2信號通路 鄰苯甲酸單(2-乙基己基)酯 細(xì)胞凋亡　出處：《沈陽醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的化學(xué)物質(zhì)低劑量效應(yīng)的利弊是近年來爭議的焦點,有研究認(rèn)為低劑量對機體存在刺激作用,是有益的;但也有持反對意見者,認(rèn)為不能忽視其產(chǎn)生的負(fù)面影響,尤其是低劑量的內(nèi)分泌干擾物,因其特殊的生物活性而倍受關(guān)注。環(huán)境內(nèi)分泌干擾物(EEDs)的研究多局限在高劑量所產(chǎn)生的毒效應(yīng)上,對環(huán)境相關(guān)劑量(低劑量)的研究尚存在不足,目前為止,認(rèn)為鄰苯二甲酸酯類對機體的生物效應(yīng)主要與其內(nèi)分泌干擾活性有關(guān)。增塑劑DEHP作為明確的內(nèi)分泌干擾物,具有多種毒性,低劑量DEHP是否會使疾病易感性增加,或存在一定的保護(hù)作用仍然不清楚。本課題選擇DEHP及其代謝產(chǎn)物MEHP作為代表物質(zhì),觀察低劑量條件下二者對細(xì)胞產(chǎn)生何種生物效應(yīng),并從細(xì)胞增殖信號通路和雌激素受體表達(dá)方面進(jìn)行機制研究,為進(jìn)一步探討內(nèi)分泌干擾物與其他疾病的關(guān)系提供更科學(xué)全面的線索。方法以雌激素受體表達(dá)陽性的人乳腺癌細(xì)胞系MCF-7作為研究對象,觀察DEHP及MEHP在不同濃度和作用時間對細(xì)胞增殖的影響,并從雌激素受體表達(dá)、功能及PGE2信號通路方面探討可能的作用機制。1.DEHP、MEHP對細(xì)胞增殖的影響。①CCK-8法及劃痕實驗檢測低劑量DEHP、MEHP對MCF-7細(xì)胞生長的影響。②細(xì)胞培養(yǎng)液中乳酸脫氫酶(LDH)活性檢測DEHP及MEHP對細(xì)胞膜是否具有損傷作用。③流式細(xì)胞儀檢測細(xì)胞凋亡情況。2.代謝產(chǎn)物MEHP對MCF-7細(xì)胞增殖影響的機制。①RT-PCR檢測雌激素受體基因表達(dá)水平的變化。②拮抗雌激素受體前后MEHP對細(xì)胞增殖及功能的影響。③檢測PGE2信號通路因子Akt、PKA、PI3K、CREB的mRNA表達(dá)。結(jié)果1.DEHP及MEHP會誘導(dǎo)MCF-7細(xì)胞增殖,其中E2及0.1μmol/LMEHP處理24h后,細(xì)胞增殖效應(yīng)顯著(P0.05),100μmol/LDEHP處理48h后,細(xì)胞生長顯著抑制(P0.05);在劃痕實驗中,E2及0.1μmol/LDEHP、0.1μmol/L MEHP處理48h后,細(xì)胞也出現(xiàn)顯著增殖(P0.05)。2.0.1 μmol/L DEHP、0.1μmol/LMEHP 處理 24h 及 48h 后,細(xì)胞培養(yǎng)液中 LDH活性均低于DMSO對照組。3.100μmol/LDEHP 處理 24h 及 48h、100μmol/LMEHP 處理 48h 后,細(xì)胞總凋亡率均顯著增高(P0.05);而E2及0.1μmol/LMEHP處理24h后,細(xì)胞總凋亡率顯著降低(P0.05),其它劑量組無明顯變化。4.1 μmol/L MEHP 處理 24h 及 48h 后,MCF-7 細(xì)胞 ERα 和 ERβ 的 mRNA 表達(dá)水平均顯著降低(P0.05)。5.拮抗雌激素受體后,因不同劑量、不同染毒時間MEHP引起的細(xì)胞增殖效應(yīng)、LDH活性降低及細(xì)胞總凋亡率降低等顯著性改變均消失。6.拮抗雌激素受體前后,MEHP引起的PGE2信號通路因子Akt、PKA、PI3K和CREB表達(dá)水平的變化情況無改變。即0.1μmol/L劑量組不同時間各因子表達(dá)水平仍分別顯著低于對照組(P0.05)。結(jié)論(1)較低劑量DEHP及MEHP會誘導(dǎo)MCF-7細(xì)胞出現(xiàn)增殖,降低凋亡率,減少對細(xì)胞膜的損傷。(2)MEHP與雌二醇具有相似的作用,雌激素受體封閉后,MEHP對MCF-7細(xì)胞產(chǎn)生的影響消失。(3)MEHP可經(jīng)雌激素受體,觸發(fā)下游PGE2信號通路,調(diào)節(jié)Akt/PI3K相關(guān)因子的表達(dá),發(fā)揮生物學(xué)作用。
[Abstract]:Objective the advantages and disadvantages of low dose effect of chemical substances have been the focus of controversy in recent years. But there are also dissenting opinions that can not be ignored its negative effects, especially low doses of endocrine disruptors. Because of its special biological activity, the study of environmental endocrine disruptor EEDsis limited to the toxic effect of high dose, but the study of environment-related dose (low dose) is still insufficient. So far, it is believed that the biological effect of phthalates on the body is mainly related to its endocrine disrupting activity. Plasticizer DEHP, as an explicit endocrine disruptor, has a variety of toxicity. It is not clear whether low dose DEHP can increase the susceptibility to disease or has some protective effect. In this study, DEHP and its metabolite MEHP were selected as representative substances. To observe the biological effect of both of them on cells at low dose, and to study the mechanism of cell proliferation signal pathway and estrogen receptor expression. To further explore the relationship between endocrine disruptors and other diseases to provide a more scientific and comprehensive clues. Methods the expression of estrogen receptor positive human breast cancer cell line MCF-7 as the study object. To observe the effect of DEHP and MEHP on cell proliferation in different concentration and time, and to explore the possible mechanism of action from the aspects of estrogen receptor expression, function and PGE2 signaling pathway. 1. Effects of MEHP on cell proliferation. 1 low dose DEHP was detected by CCK-8 method and scratch test. Effects of MEHP on the growth of MCF-7 cells. Whether DEHP and MEHP have damage to cell membrane. 3. Flow cytometry is used to detect apoptosis. 2. The mechanism of the effect of metabolite MEHP on the proliferation of MCF-7 cells. The changes of estrogen receptor gene expression were detected by -PCR. 2. The effect of MEHP on cell proliferation and function before and after estrogen receptor was antagonized. 3. The PGE2 signal pathway factor (Akt) was detected. Results 1. DEHP and MEHP could induce the proliferation of MCF-7 cells. After treatment with E _ 2 and 0.1 渭 mol/LMEHP for 24 h, the proliferation effect of E _ 2 and 0.1 渭 mol/LMEHP was significantly increased after 48 h treatment with 100 渭 mol/LDEHP. Cell growth was significantly inhibited by P0.05; In the scratch test, E2 and 0.1 渭 mol / L DEHPX 0.1 渭 mol/L MEHP were treated for 48 h. Cell proliferation was also observed after 24 and 48 h treatment with P0.05, 2.0.1 渭 mol/L DEHP0.1 渭 mol/LMEHP. The activity of LDH in the cell culture medium was lower than that in the DMSO control group. 3.100 渭 mol/LDEHP for 24 h and 48 h. After treatment with 100 渭 mol/LMEHP for 48 h, the total apoptotic rate increased significantly (P 0.05). After treatment with E2 and 0.1 渭 mol/LMEHP for 24 h, the total apoptosis rate decreased significantly (P 0.05). There was no significant change in other dose groups. 4. 1 渭 mol/L MEHP for 24 h and 48 h. The mRNA expression of ER 偽 and ER 尾 in MCF-7 cells was significantly decreased after antagonizing estrogen receptor in different doses. The significant changes of MEHP induced by different exposure time, such as the decrease of cell proliferation activity and the decrease of total apoptosis rate, disappeared. 6. Before and after antagonizing estrogen receptor. MEHP induced PGE2 signal pathway factor Akton-PKA. The expression levels of PI3K and CREB were not changed, that is, the expression level of each factor in 0.1 渭 mol/L group was still significantly lower than that in control group at different time. Lower doses of DEHP and MEHP could induce the proliferation of MCF-7 cells. Reduce apoptosis rate, reduce cell membrane damage. MEHP and estradiol have similar effects, estrogen receptor blocking. The effect of MEHP on the production of MCF-7 cells disappeared. The estrogen receptor could trigger the downstream PGE2 signaling pathway and regulate the expression of Akt/PI3K related factors. Play a biological role.
【學(xué)位授予單位】：沈陽醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

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