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  本文選題：雙酚A　切入點：遺傳毒性　出處：《蘇州大學》2013年碩士論文　論文類型：學位論文

【摘要】：目的：雙酚A（bisphenol A, BPA），是一種廣泛使用的化工原料，添加BPA可使塑料制品具有輕巧耐用、無色透明以及加強抗沖擊性等特點，并有效防止水果和蔬菜從內(nèi)部侵蝕金屬容器，所以BPA成為制造聚碳酸酯、環(huán)氧樹脂、食品包裝、牙科填充劑、食品罐頭涂料和醫(yī)療器械等產(chǎn)品的重要原料。BPA具有毒作用劑量小、潛伏時間長等特點，對健康造成多種潛在危害，國內(nèi)外許多學者對BPA的毒性作用進行了研究報道。目前對BPA的毒性研究多集中在內(nèi)分泌系統(tǒng)、生殖系統(tǒng)、神經(jīng)系統(tǒng)等方面，其遺傳毒性還有待進一步研究。本實驗通過體外染毒檢測BPA對中國倉鼠卵巢細胞（CHO細胞）的細胞存活率、DNA的直接損傷作用、微核率以及染色體畸變率，并通過Ames試驗檢測BPA的致突變性，通過將上述檢測不同遺傳學終點的試驗相結(jié)合探討B(tài)PA的遺傳毒性。 方法：(1)以倉鼠卵巢細胞（CHO細胞）作為實驗系統(tǒng)，選用四甲基偶氮唑鹽法（即MTT比色法）檢測不同濃度雙酚A對CHO細胞作用不同時間后，對細胞存活率的影響；（2）采用單細胞凝膠電泳試驗（即SCGE彗星試驗），了解不同濃度及不同作用時間下雙酚A對CHO細胞DNA單鏈斷裂的損傷程度；（3）采用微核試驗研究不同濃度雙酚A對CHO細胞微核率的影響；（4）采用染色體畸變分析檢測不同濃度雙酚A對CHO細胞染色體畸變率及誘導的主要畸變類型；（5）通過Ames試驗了解不同濃度雙酚A的致突變效應。 結(jié)果：（1）MTT試驗結(jié)果顯示，分別經(jīng)12h及24h染毒處理后，當雙酚A終濃度為40μmol/L時，CHO細胞的存活率顯著高于陰性對照組。當濃度高于80μmol/L時，，細胞存活率呈現(xiàn)下降趨勢，各染毒組吸光度值與陰性對照組相比均具有統(tǒng)計學差異。（2）單細胞凝膠電泳實驗結(jié)果顯示，經(jīng)12h及24h處理后，雙酚A濃度高于80μmol/L時，可引起CHO細胞的拖尾率、尾部DNA百分含量及尾長增加。（3）微核試驗和染色體畸變試驗表明，在雙酚A處理24h后，當濃度高于100μmol/L時，CHO細胞的微核率和染色體畸變率顯著增加。其中染色體畸變的類型以斷裂、無著絲粒斷片和裂隙為主。（4）Ames試驗檢測的BPA染毒濃度為10μg/皿～5mg/皿共九個劑量組，其結(jié)果為陰性。在活化和非活化條件下，與陰性對照組比較，可見部分菌株的回變菌落數(shù)有所增加，但并無統(tǒng)計學差異。 結(jié)論:（1）外培養(yǎng)條件下，雙酚A在染毒濃度為40μmol/L作用12h后，可顯著刺激細胞的生長，表現(xiàn)出一定的刺激效應。從80μmol/L開始，染毒12h對CHO細胞的增殖有明顯的抑制作用，表現(xiàn)出細胞毒性作用。（2）雙酚A可引起CHO細胞的拖尾率、尾部DNA百分含量、尾長及Olive尾矩增加。提示雙酚A可引起體外培養(yǎng)的細胞產(chǎn)生DNA的單鏈損傷。（3）雙酚A能顯著增加CHO細胞的微核率和染色體畸變率。其中染色體畸變的類型以斷裂、無著絲粒斷片和裂隙為主。（4）Ames試驗未檢測到雙酚A的致基因突變作用。在活化和非活化條件下，與陰性對照組相比，可見部分菌株的回變菌落數(shù)有增加趨勢，但并無統(tǒng)計學意義。提示雙酚A可能并不存在致基因突變的作用。
[Abstract]:Objective: bisphenol A (bisphenol A BPA), is a widely used chemical raw materials, adding BPA can make plastic products is lightweight and durable, colorless and transparent and strengthening the impact resistance, and effectively prevent the erosion of metal containers of fruit and vegetables from the inside, so BPA is manufacturing polycarbonate, epoxy resin, food packaging. Dental filler,.BPA important raw material of canned food coatings and medical equipment and other products with the toxic effect of small dosage, the characteristics of the potential for a long time, causing a variety of potential health hazards and toxic effects of many domestic and foreign scholars have conducted the research to the BPA report. The present study toxicity of BPA mainly focus on the endocrine system, reproductive system. The neural system, the genetic toxicity remains to be further studied. The experiments of in vitro detection of BPA China hamster ovary cells (CHO cells) cell viability, direct injury DNA The micronucleus rate and chromosome aberration rate were detected. The mutagenicity of BPA was detected by Ames test. The genotoxicity of BPA was investigated by combining the above test of different genetic endpoints.
Methods: (1) in Chinese hamster ovary cells (CHO cells) as an experimental system, using four methyl thiazolyl tetrazolium method (MTT assay) to detect different concentrations of bisphenol A on CHO cells for different time, the rate of cell survival; (2) using single cell gel electrophoresis assay (comet SCGE to understand the extent of damage test), different concentrations and different action time of bisphenol A on CHO cells of DNA single strand breaks; (3) the experimental study on Micronucleus of different concentrations of bisphenol A on the effects of micronucleus rate of CHO cells; (4) by chromosome aberration detection and analysis of different concentration of bisphenol A on the main CHO cell chromosome aberration and distortion induction type; (5) to understand the mutagenic effects of different concentrations of bisphenol A by Ames test.
Results: (1) MTT test results showed that 12h and 24h respectively after exposure after treatment, when the final concentration of bisphenol A is 40 mol/L, the survival rate of CHO cells was significantly higher than that of the negative control group. When the concentration is higher than 80 mol/L, the cell survival rate decreased, the absorbance values were compared with the exposure group significant difference with the negative control group. (2) experimental results of single cell gel electrophoresis showed that the 12h and 24h after treatment, the concentration of bisphenol A is higher than 80 mol/L, can cause CHO cell comet frequency, tail DNA% and tail length increased. (3) showed that micronucleus test and chromosome aberration test in bisphenol A after 24h treatment, when the concentration is higher than 100 mol/L, CHO cell micronucleus rate and chromosome aberration rate increased significantly. The type of chromosome aberration to fracture, acentric fragment and fissures. (4) the exposure concentration of BPA Ames test for 10 g/ ~ nine 5mg/ dish dish In the dose group, the results were negative. When activated and non activated, compared with the negative control group, the number of the colony of some strains increased, but there was no statistical difference.
Conclusion: (1) the culture conditions, bisphenol A exposure concentrations in 40 mol/L after 12h treatment can significantly stimulate cell growth, showed some stimulation effect. From the beginning of the 80 mol/L 12h exposure on the proliferation of CHO cells was significantly inhibited and showed cytotoxicity (2.) bisphenol A can cause CHO cell comet frequency, tail DNA%, tail length and Olive tail moment increased. Single strand damage that bisphenol A can cause the cells to produce DNA. (3) bisphenol A could significantly increase CHO cell micronucleus rate and chromosome aberration rate of chromosome aberration in the type. Fracture, acentric fragment and fissures. (4) Ames test did not detect mutagenic effects of bisphenol A. In the activation and non activation conditions, compared with the negative control group, visible strain change counts increased, but there was no statistically significant. Bisphenol A may prompt There is no effect of gene mutation.

【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114

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