低剂量邻苯二甲酸二乙基己酯对MCF-7细胞增殖的影响及机制研究

发布时间：2018-01-28 09:03

本文关键词： 低剂量邻苯二甲酸二乙基己酯人乳腺癌细胞MCF-7 PGE_2信号通路邻苯甲酸单(2-乙基己基)酯细胞凋亡　出处：《沈阳医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的化学物质低剂量效应的利弊是近年来争议的焦点,有研究认为低剂量对机体存在刺激作用,是有益的;但也有持反对意见者,认为不能忽视其产生的负面影响,尤其是低剂量的内分泌干扰物,因其特殊的生物活性而倍受关注。环境内分泌干扰物(EEDs)的研究多局限在高剂量所产生的毒效应上,对环境相关剂量(低剂量)的研究尚存在不足,目前为止,认为邻苯二甲酸酯类对机体的生物效应主要与其内分泌干扰活性有关。增塑剂DEHP作为明确的内分泌干扰物,具有多种毒性,低剂量DEHP是否会使疾病易感性增加,或存在一定的保护作用仍然不清楚。本课题选择DEHP及其代谢产物MEHP作为代表物质,观察低剂量条件下二者对细胞产生何种生物效应,并从细胞增殖信号通路和雌激素受体表达方面进行机制研究,为进一步探讨内分泌干扰物与其他疾病的关系提供更科学全面的线索。方法以雌激素受体表达阳性的人乳腺癌细胞系MCF-7作为研究对象,观察DEHP及MEHP在不同浓度和作用时间对细胞增殖的影响,并从雌激素受体表达、功能及PGE2信号通路方面探讨可能的作用机制。1.DEHP、MEHP对细胞增殖的影响。①CCK-8法及划痕实验检测低剂量DEHP、MEHP对MCF-7细胞生长的影响。②细胞培养液中乳酸脱氢酶(LDH)活性检测DEHP及MEHP对细胞膜是否具有损伤作用。③流式细胞仪检测细胞凋亡情况。2.代谢产物MEHP对MCF-7细胞增殖影响的机制。①RT-PCR检测雌激素受体基因表达水平的变化。②拮抗雌激素受体前后MEHP对细胞增殖及功能的影响。③检测PGE2信号通路因子Akt、PKA、PI3K、CREB的mRNA表达。结果1.DEHP及MEHP会诱导MCF-7细胞增殖,其中E2及0.1μmol/LMEHP处理24h后,细胞增殖效应显著(P0.05),100μmol/LDEHP处理48h后,细胞生长显著抑制(P0.05);在划痕实验中,E2及0.1μmol/LDEHP、0.1μmol/L MEHP处理48h后,细胞也出现显著增殖(P0.05)。2.0.1 μmol/L DEHP、0.1μmol/LMEHP 处理 24h 及 48h 后,细胞培养液中 LDH活性均低于DMSO对照组。3.100μmol/LDEHP 处理 24h 及 48h、100μmol/LMEHP 处理 48h 后,细胞总凋亡率均显著增高(P0.05);而E2及0.1μmol/LMEHP处理24h后,细胞总凋亡率显著降低(P0.05),其它剂量组无明显变化。4.1 μmol/L MEHP 处理 24h 及 48h 后,MCF-7 细胞 ERα 和 ERβ 的 mRNA 表达水平均显著降低(P0.05)。5.拮抗雌激素受体后,因不同剂量、不同染毒时间MEHP引起的细胞增殖效应、LDH活性降低及细胞总凋亡率降低等显著性改变均消失。6.拮抗雌激素受体前后,MEHP引起的PGE2信号通路因子Akt、PKA、PI3K和CREB表达水平的变化情况无改变。即0.1μmol/L剂量组不同时间各因子表达水平仍分别显著低于对照组(P0.05)。结论(1)较低剂量DEHP及MEHP会诱导MCF-7细胞出现增殖,降低凋亡率,减少对细胞膜的损伤。(2)MEHP与雌二醇具有相似的作用,雌激素受体封闭后,MEHP对MCF-7细胞产生的影响消失。(3)MEHP可经雌激素受体,触发下游PGE2信号通路,调节Akt/PI3K相关因子的表达,发挥生物学作用。
[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 伪 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.
【学位授予单位】：沈阳医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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