雙酚A、對枯基苯酚和己烯雌酚對MCF-7細胞的單一和聯(lián)合效應(yīng)研究
發(fā)布時間：2019-02-15 02:58
【摘要】：隨著人類社會農(nóng)業(yè)、工業(yè)、經(jīng)濟和科學的快速發(fā)展,大量化合物被合成并通過各種遷移轉(zhuǎn)化途徑進入環(huán)境中。外源雌激素(Xenoestrogens, XEs)就是其中的一大類物質(zhì),它可以通過模擬或者干擾內(nèi)源雌激素的作用方式而發(fā)揮雌激素效應(yīng)。不僅僅在水體,大氣和土壤等環(huán)境介質(zhì)中能檢測到XEs,它也廣泛存在于各種動物體內(nèi)甚至是人體中,人類時時刻刻都進行著多種XEs的聯(lián)合暴露。與此同時,大量研究證實了XEs暴露與乳腺癌、子宮內(nèi)膜癌、卵巢癌、宮頸癌以及前列腺癌等癌癥的發(fā)生發(fā)展有著密切關(guān)系。本論文以雙酚A (BPA),己烯雌酚(DES)和對枯基苯酚(4-CP)三種結(jié)構(gòu)類似的XEs為目標化合物,以雌激素受體陽性的人類乳腺癌MCF-7細胞為受試對象,通過細胞增殖實驗研究了三種目標化合物和人體濃度的雌二醇(E2)單獨作用和聯(lián)合作用的雌激素效應(yīng)。采用蛋白質(zhì)免疫印跡法(Western blot)檢測了雌激素單獨和聯(lián)合作用時雌激素受體α (ERα)、雌激素相關(guān)受體γ (ERRγ)和抗凋亡蛋白Bcl-2的表達水平,推測引起細胞增殖可能的機制。得到以下的研究結(jié)果：(1) BPA、4-CP、DES在誘導(dǎo)細胞增殖時具有時間依賴性和低劑量、非線性效應(yīng)特征。三種XEs誘導(dǎo)細胞增殖能力為DESBPA4-CP,而高濃度時的細胞毒性為DES4-CPBPA。(2)三種目標化合物都可以通過與ERa結(jié)合,激活基因組通路,從而影響細胞的生長和增殖。四種雌激素與ERa結(jié)合能力為：E2 DES 4-CP BPA。 ERRγ在三種外源雌激素引起的增值效應(yīng)中也具有重要作用,BPA和4-CP對ERRγ的親和力大于DES,而DES大于E2。在單一效應(yīng)實驗中,Bcl-2蛋白的表達與各實驗組的細胞增殖率之間有比較好的對應(yīng)關(guān)系,提示：三種XEs也可以通過作用于細胞凋亡蛋白家族,從而對細胞增殖和凋亡產(chǎn)生影響。(3)三種外源雌激素兩兩混合時,BPA和DES可以通過混合作用相互促進,發(fā)揮出更強的雌激素效應(yīng)。這可能是由于摻入BPA后,混合物的ERRγ的表達水平相對于DES單獨作用時更高；而摻入DES后,混合物結(jié)合ERa的水平相對于DES單獨作用時更強。但對于三種外源雌激素形成的三元混合物,其增值效應(yīng)有一定減弱,高濃度的4-CP會使混合物發(fā)揮抑制增殖的效應(yīng),而相應(yīng)的Western blot結(jié)果顯示XEs三元混合對ERα、 ERRγ和Bcl-2的表達均有不同程度的影響。(4)生理濃度的E2可以使單一XEs對MCF-7細胞的促增殖能力顯著提高。產(chǎn)生這一現(xiàn)象的原因可能是由于摻入XEs后,混合物的ERRγ的表達水平相對于E2單獨作用時更高；而摻入E2后,混合物結(jié)合ERa的水平相對于XEs單獨作用時更強。相反地,當E2與兩種或三種XEs混合時,E2會弱化XEs混合物產(chǎn)生的雌激素效應(yīng),很多實驗組雌激素活性基本喪失。且其對ERα、 ERRγ和Bcl-2表達的影響并不具有一般規(guī)律性。
[Abstract]:With the rapid development of agriculture, industry, economy and science, a large number of compounds have been synthesized and entered into the environment through various migration and transformation pathways. Exogenous estrogen (Xenoestrogens, XEs) is one of the major substances. It can play an estrogen effect by simulating or interfering with the action of endogenous estrogen. Not only can XEs, be detected in environmental media such as water body, atmosphere and soil, but it also exists in various animals and even in human body. Human beings are exposed to a variety of XEs all the time. At the same time, numerous studies have confirmed that XEs exposure is closely related to the occurrence and development of breast, endometrial, ovarian, cervical and prostate cancers. In this study, three similar XEs compounds, bisphenol A (BPA), diethylstilbestrol (DES) and p-withered phenol (4-CP), were used as target compounds, and MCF-7 cells with positive estrogen receptor were used as subjects. The estrogenic effects of three target compounds and estradiol (E _ 2) were studied by cell proliferation experiments. The expression levels of estrogen receptor 偽 (ER 偽), estrogen associated receptor 緯 (ERR 緯) and anti-apoptotic protein (Bcl-2) were detected by Western blot (Western blot). We speculate on the possible mechanism of cell proliferation. The results are as follows: (1) BPA,4-CP,DES has the characteristics of time-dependent, low-dose and nonlinear effects in the induction of cell proliferation. Three kinds of XEs can induce cell proliferation ability to be DESBPA4-CP, but at high concentration the cytotoxicity is DES4-CPBPA. (2). All three target compounds can activate genomic pathway by binding with ERa, thus affecting cell growth and proliferation. The binding ability of four estrogens to ERa is E2 DES 4-CP BPA.. ERR 緯 also plays an important role in the three exogenous estrogen-induced value-added effects. The affinity of BPA and 4-CP to ERR 緯 is greater than that of DES, and DES is greater than E2. In the single effect experiment, the expression of Bcl-2 protein had a good correspondence with the cell proliferation rate of each experimental group, which suggested that three kinds of XEs could also act on the apoptotic protein family. Thus, the proliferation and apoptosis of cells were affected. (3) when the three exogenous estrogens were mixed, BPA and DES could promote each other and play a stronger estrogen effect. This may be due to the higher expression level of ERR 緯 in the mixture after incorporation of BPA than that in the DES alone, while the level of ERa binding in the mixture with DES is stronger than that with DES alone. However, for the ternary mixture of three exogenous estrogens, the increment effect was weakened, and the high concentration of 4-CP could inhibit the proliferation of the mixture, and the corresponding Western blot results showed that the XEs ternary mixture could inhibit the proliferation of ER 偽. The expression of ERR 緯 and Bcl-2 were affected in different degrees. (4) the physiological concentration of E2 could significantly increase the proliferation of MCF-7 cells induced by single XEs. The reason for this phenomenon may be that the expression level of ERR 緯 in mixture is higher than that in E2 alone, and the level of ERa binding in mixture is stronger than that in XEs alone. On the contrary, when E2 was mixed with two or three kinds of XEs, E2 weakened the estrogenic effect of XEs mixture, and the estrogenic activity of many experimental groups was basically lost. Moreover, its effect on the expression of ER 偽, ERR 緯 and Bcl-2 has no general regularity.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：X503
本文編號：2422890
[Abstract]:With the rapid development of agriculture, industry, economy and science, a large number of compounds have been synthesized and entered into the environment through various migration and transformation pathways. Exogenous estrogen (Xenoestrogens, XEs) is one of the major substances. It can play an estrogen effect by simulating or interfering with the action of endogenous estrogen. Not only can XEs, be detected in environmental media such as water body, atmosphere and soil, but it also exists in various animals and even in human body. Human beings are exposed to a variety of XEs all the time. At the same time, numerous studies have confirmed that XEs exposure is closely related to the occurrence and development of breast, endometrial, ovarian, cervical and prostate cancers. In this study, three similar XEs compounds, bisphenol A (BPA), diethylstilbestrol (DES) and p-withered phenol (4-CP), were used as target compounds, and MCF-7 cells with positive estrogen receptor were used as subjects. The estrogenic effects of three target compounds and estradiol (E _ 2) were studied by cell proliferation experiments. The expression levels of estrogen receptor 偽 (ER 偽), estrogen associated receptor 緯 (ERR 緯) and anti-apoptotic protein (Bcl-2) were detected by Western blot (Western blot). We speculate on the possible mechanism of cell proliferation. The results are as follows: (1) BPA,4-CP,DES has the characteristics of time-dependent, low-dose and nonlinear effects in the induction of cell proliferation. Three kinds of XEs can induce cell proliferation ability to be DESBPA4-CP, but at high concentration the cytotoxicity is DES4-CPBPA. (2). All three target compounds can activate genomic pathway by binding with ERa, thus affecting cell growth and proliferation. The binding ability of four estrogens to ERa is E2 DES 4-CP BPA.. ERR 緯 also plays an important role in the three exogenous estrogen-induced value-added effects. The affinity of BPA and 4-CP to ERR 緯 is greater than that of DES, and DES is greater than E2. In the single effect experiment, the expression of Bcl-2 protein had a good correspondence with the cell proliferation rate of each experimental group, which suggested that three kinds of XEs could also act on the apoptotic protein family. Thus, the proliferation and apoptosis of cells were affected. (3) when the three exogenous estrogens were mixed, BPA and DES could promote each other and play a stronger estrogen effect. This may be due to the higher expression level of ERR 緯 in the mixture after incorporation of BPA than that in the DES alone, while the level of ERa binding in the mixture with DES is stronger than that with DES alone. However, for the ternary mixture of three exogenous estrogens, the increment effect was weakened, and the high concentration of 4-CP could inhibit the proliferation of the mixture, and the corresponding Western blot results showed that the XEs ternary mixture could inhibit the proliferation of ER 偽. The expression of ERR 緯 and Bcl-2 were affected in different degrees. (4) the physiological concentration of E2 could significantly increase the proliferation of MCF-7 cells induced by single XEs. The reason for this phenomenon may be that the expression level of ERR 緯 in mixture is higher than that in E2 alone, and the level of ERa binding in mixture is stronger than that in XEs alone. On the contrary, when E2 was mixed with two or three kinds of XEs, E2 weakened the estrogenic effect of XEs mixture, and the estrogenic activity of many experimental groups was basically lost. Moreover, its effect on the expression of ER 偽, ERR 緯 and Bcl-2 has no general regularity.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：X503
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