本文選題：雙酚A類化合物 + 微板毒性分析��； 參考：《西南大學(xué)》2012年碩士論文

【摘要】：雙酚A(BPA)是廣泛存在的環(huán)境內(nèi)分泌干擾物(EDCs), BPA類化合物作為環(huán)氧樹脂,聚碳酸酯塑料以及阻燃劑等產(chǎn)品添加劑,在水杯、奶瓶、食品飲料容器、醫(yī)療設(shè)備等生產(chǎn)中被廣泛使用,并被不斷開發(fā)做新的用途。這些制品在長期使用或在高溫、堿性條件下,BPA都會緩慢釋放到周圍環(huán)境中,并且很容易在環(huán)境中積累,并通過用水,空氣,土壤和食品污染等對周圍生物產(chǎn)生影響。 BPA類化合物的雌激效應(yīng)一直是研究的重點,但是關(guān)于其化合物毒性效應(yīng)的研究較少。到目前為止,研究發(fā)現(xiàn)BPA在較窄的濃度范圍內(nèi)對MCF-7的損傷作用主要依賴于類雌激素作用干擾胞內(nèi)酶活性及細胞代謝活性影響細胞增殖,而微高濃度的BPA會對細胞造成損傷,導(dǎo)致細胞凋亡。因此在細胞水平上研究BPA類化合物的毒性效應(yīng)具有重要的意義。同時,環(huán)境中的污染物并不是單獨存在的,人群在生產(chǎn)生活中總是處于各種污染物的混合暴露之下。研究污染物的聯(lián)合作用比污染物的單獨作用更加貼近實際,更有現(xiàn)實意義。本實驗主要是通過微板毒性分析法研究8種BPA類化合物對雌激素受體缺失MCF-7細胞的毒性效應(yīng),并初步結(jié)合DNA損傷以及細胞膜損傷研究為進一步評價BPA類化合物的環(huán)境毒性效應(yīng)奠定基礎(chǔ)。 實驗選用8種雙酚A類化合物,包括2,2-雙(4-羥基苯基)丙烷(簡稱BPA),2,2-二(4-羥基苯基)丁烷(簡稱BPB),4,4'-(1-甲基亞乙基)雙(2-甲基苯甲醚)(簡稱BPC),4,4'-亞乙基雙苯酚(簡稱BPE),2,2-雙(2-羥基-5-聯(lián)苯基)丙烷(簡稱BPQ,4,4'-硫代二苯酚(TDP),雙酚AF(簡稱BPAF),4,4'-(1-苯乙基)雙酚(簡稱BPAP),采用四唑鹽(MTS/PMS)比色法檢測此8種BPA類化合物和12種根據(jù)等效應(yīng)濃度比和均勻濃度比設(shè)計的混合物對雌激素受體缺失人乳腺癌細胞(MCF-7(ER-))增殖活性的影響,用非線性最小二乘法擬合得到濃度-效應(yīng)曲線(CRC),以濃度加和模型(CA)和獨立作用模型(IA)為參考模型,研究混合物的相互作用規(guī)律。通過2,4一二硝基苯肼法檢測8種雙酚A類化合物對MCF-7(ER-)細胞乳酸脫氫酶(LDH)露出率的影響,單細胞凝膠電泳(SCGE)檢測了5種雙酚A類化合物對MCF-7(ER-)細胞DNA損傷。 結(jié)果發(fā)現(xiàn)所有的劑量-效應(yīng)曲線(CRC)均能用Weibull或者Logit函數(shù)有效表征,以模型估算的半數(shù)效應(yīng)濃度負對數(shù)值(pEC50)評估8種化合物的毒性大小依次為：BPQBPAFBPAPBPBBPCTDPBPE BPA;應(yīng)用劑量加和(CA)模型和獨立作用(IA)模型對混合物毒性進行評估與預(yù)測,結(jié)果8種BPA類化合物各種濃度組合的混合物毒性均可用CA模型有效預(yù)測；LDH檢測以及SCGE檢測BPA類化合物對MCF-7(ER-)的損傷作用,首先是干擾酶活性及微量元素代謝,抑制細胞增殖,然后是對細胞核DNA造成損傷,同時細胞膜也受到損傷,細胞開始出現(xiàn)大量壞死。 實驗最后對構(gòu)建穩(wěn)定表達Metridia熒光素酶的MCF-7(ER-)細胞毒性檢測系統(tǒng)進行探索,旨在通過測定熒光素酶活性間接反映環(huán)境污染物的毒性作用,使得毒性檢測更加直觀易行。
[Abstract]:Bisphenol A (BPA) is a widespread environmental endocrine disruptor, BPA compounds are widely used as additives for epoxy resin, polycarbonate plastics and flame retardants in the production of water cups, milk bottles, food and beverage containers, medical equipment, etc.And has been constantly developed for new uses.These products can be released slowly into the surrounding environment under the condition of long term use or high temperature and alkalinity, and can easily accumulate in the environment, and influence the surrounding organisms through water, air, soil and food pollution.The female excitatory effect of BPA compounds has been the focus of study, but the toxicity of its compounds is less studied.Up to now, it has been found that the damage of BPA to MCF-7 in a narrow concentration range mainly depends on the estrogenic effect on the interference of intracellular enzyme activity and cell metabolic activity, while the slightly high concentration of BPA can damage the cells.Leading to apoptosis.Therefore, it is of great significance to study the toxic effects of BPA compounds at the cellular level.At the same time, environmental pollutants do not exist alone, people are always exposed to a mixture of pollutants in production and life.It is more practical and meaningful to study the combined action of pollutants than the single action of pollutants.In this study, microplate toxicity assay was used to study the toxic effects of eight BPA compounds on estrogen receptor deficient MCF-7 cells.Combined with the study of DNA damage and cell membrane damage, the study laid a foundation for further evaluation of the environmental toxicity of BPA compounds.Eight kinds of bisphenol A compounds were used in the experiment.Including BPA-2-Bis 4- hydroxyphenyl) propane (abbreviated as BPBX 4H 4H 4H 4N) BPCO 4H 4N 4 ethylphenyl bisphenol (abbreviated as BPE2O2O2O2-HYDROXY5HYDIPHENYL) propane (abbreviated as BPCO 4HYDROXYLPHENYL) propane (abbreviated as BPCO 4HYDROXYLPHETHYL) propane (BPCO 4HYDROXYLPHENYL for short for short for BPCO 4HYDROXYLPHENYL)Eight BPA compounds and 12 mixtures designed according to the equal effect concentration ratio and uniform concentration ratio were determined by using the method of tetrazolium salt MTS / PMS-colorimetry to detect the effects of eight BPA compounds and 12 mixtures designed according to the equal effect concentration ratio and the uniform concentration ratio, respectively, and bisphenol A F (abbreviated as BPA PAPP) to determine the estrogenicity of the eight BPA compounds and 12 mixtures designed according to the equal effect concentration ratio and the uniform concentration ratio.The effect of deletion of human breast cancer cell line MCF-7 ER-G) on proliferative activity,The concentration-effect curve was obtained by using the nonlinear least square method. The interaction law of the mixture was studied by using the concentration addition model (CAA) and the independent interaction model (IAA) as reference models.The effects of eight bisphenol A compounds on the exposure rate of lactate dehydrogenase (LDHs) in MCF-7ER-cells were detected by 2O4-dinitrophenylhydrazine method. Single cell gel electrophoresis (SCGE) was used to detect the DNA damage of MCF-7ER-cells induced by five bisphenol A compounds.The results show that all dose-response curves can be effectively characterized by Weibull or Logit functions.The toxicity of the eight compounds was evaluated in order of: BPQBPAFBPAPBPBBPCTDPE BPAs, and the toxicity of the mixture was evaluated and predicted by the dose-add-sum model and the independent action model.Results the toxicity of the mixture of 8 kinds of BPA compounds with different concentrations can be predicted by CA model and SCGE. The first step is to interfere with enzyme activity and trace element metabolism and inhibit cell proliferation.Then the nuclear DNA was damaged, the cell membrane was also damaged, and the cell began to suffer a lot of necrosis.At the end of the experiment, we explored the construction of MCF-7 ER-ER-C cytotoxicity detection system which stably expressed Metridia luciferase, in order to reflect the toxicity of environmental pollutants indirectly by measuring luciferase activity, making the toxicity detection more intuitionistic and practical.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114

【參考文獻】

相關(guān)期刊論文 前10條

1 楊麗平;趙敬湘;李露斯;袁紅峰;劉大慶;王蘊芳;南雪;白慈賢;裴雪濤;;穩(wěn)定表達螢火蟲熒光素酶的9L~(luc)細胞株的構(gòu)建[J];重慶醫(yī)學(xué);2007年09期

2 趙海濤,張?zhí)鞂?朱江波,朱玉平,萬旭英,王征,馬璽里;基于熒光素酶報告基因的MCF7細胞雌激素樣物質(zhì)檢測方法的建立[J];第二軍醫(yī)大學(xué)學(xué)報;2005年09期

3 張志仁,徐順清,周宜開,任恕,劉志偉;蟲熒光素酶報告基因用于二VA英類化學(xué)物質(zhì)的檢測[J];分析化學(xué);2001年07期

4 高蕾,朱志忠;國際雙酚A市場現(xiàn)狀及我國發(fā)展前景[J];化工技術(shù)經(jīng)濟;2004年01期

5 肖菊;劉樹深;高音旋;張亞雷;;均勻設(shè)計用于研究硝基苯衍生物對青海弧菌Q67的聯(lián)合毒性[J];環(huán)境科學(xué)研究;2008年02期

6 徐挺,孔繁翔,孫成,談健康,韓小波,閻榮;基因重組細胞在環(huán)境樣品多氯聯(lián)苯檢測中的應(yīng)用[J];環(huán)境科學(xué);2004年01期

7 李湘鳴;羅方妮;陳春波;;重組酵母細胞快速篩選環(huán)境雌激素系統(tǒng)的建立[J];環(huán)境與健康雜志;2006年02期

8 陰海鵬;張玲;李翠玲;顧洪濤;劉文敏;許強;趙勇;;雙酚A對人卵巢癌3AO細胞、前列腺癌DU145細胞及子宮內(nèi)膜癌HHUA細胞增殖活性的影響[J];環(huán)境與健康雜志;2011年01期

9 沈惠云,徐培渝,余文三,劉玉清,吳德生;對-壬基酚對MCF-7人類乳腺癌細胞雌激素受體表達的影響[J];四川大學(xué)學(xué)報(醫(yī)學(xué)版);2003年04期

10 張江華,李華文,石丹,江明,郝巧玲,周宜開,呂斌;雙酚A對人胚肝細胞DNA損傷和修復(fù)功能的影響[J];環(huán)境與職業(yè)醫(yī)學(xué);2005年03期

，

本文編號：1761287