【摘要】：環(huán)境雌激素雙酚A（Bisphenol A，BPA）是一種重要的化工原料，其廣泛的應(yīng)用范圍導致了無處不在的暴露。BPA具有弱雌激素樣作用和強抗雄激素樣作用，可對雄性生殖系統(tǒng)產(chǎn)生影響。然而關(guān)于BPA雄性生殖毒性的人群研究和相關(guān)機制研究的報道極為有限。本研究通過人群研究探討B(tài)PA對男性的生殖毒性，通過動物實驗研究進一步探索可能的機制，為明確BPA的雄性生殖毒性及其機理提供科學依據(jù)。 目的 采用人群研究和動物實驗研究相結(jié)合的方法，探討B(tài)PA暴露對男性生殖功能的影響，并進一步探索可能的機制，為BPA對生殖功能的作用及其機制的研究提供新的思路。 1．通過分析BPA對男性精子質(zhì)量、血清性激素水平以及性功能之間的關(guān)系，探討B(tài)PA對男性的生殖毒性； 2．探討B(tài)PA對雄性大鼠生殖系統(tǒng)及精子質(zhì)量的影響，，以及對主要激素睪酮（T）與卵泡刺激素（FSH）的影響； 3．通過研究BPA對激素受體AR、FSHR和信號通路相關(guān)基因Src、ERK1/2、CREB的影響，初步探討B(tài)PA在精子發(fā)生過程中可能的分子機制。 方法 人群研究：收集543名研究對象，采用結(jié)構(gòu)式問卷調(diào)查研究對象一般人口學特征、既往病史及用藥史、生活方式、性生活史、BPA暴露史等。采集研究對象尿液、精液、血液，應(yīng)用高效液相色譜法(HPLC)檢測尿液中BPA水平；計算機輔助精子分析系統(tǒng)檢測精子數(shù)量、活動力和存活率，改良巴氏染色法檢測精子形態(tài)學；放射免疫法(RIA)檢測血清卵泡刺激素（FSH）、泌乳素（PRL）、睪酮（T）、雌二醇（E2）水平，酶聯(lián)免疫吸附法(ELISA)檢測血清抑制素B (INB)、游離睪酮（FT）、性激素結(jié)合球蛋白（SHBG）和雄烯二酮（AD）水平。應(yīng)用SPSS16.0軟件建立數(shù)據(jù)庫并運用t檢驗、χ~2檢驗、趨勢χ~2檢驗、pearson相關(guān)分析、spearman秩相關(guān)分析、多元線性回歸分析等檢驗方法進行統(tǒng)計分析。 動物實驗：雄性Wistar大鼠（200±20g）28只按體重隨機分為4組，每天固定時間灌胃染毒，染毒劑量分別為BPA0、50、100、200mg/（kg.d），連續(xù)4周后處死大鼠，采集生殖系統(tǒng)臟器、血清及一側(cè)附睪尾精子。計算機輔助分析檢測大鼠精子密度、活力、存活率，改良巴氏染色法檢測精子畸形率；放射免疫法檢測血清T、FSH水平。采用Real-time PCR和Western-blot法分別檢測大鼠睪丸中AR、FSHR、Src、ERK1/2、CREB基因mRNA和蛋白表達水平。采用SPSS16.0建立數(shù)據(jù)庫并運用單因素方差分析、Dunnet-t檢驗進行統(tǒng)計分析。 結(jié)果 人群研究 1．尿液中BPA濃度升高與精子密度低下、精子總數(shù)低下、精子活動力低下、精子存活率低下之間存在劑量效應(yīng)關(guān)系；在調(diào)整可疑混雜因素后，尿液中BPA濃度與精子密度、總數(shù)、活動力、存活率之間存在負相關(guān)關(guān)系。 2．尿液BPA濃度升高與FT, AD, SHBG, PRL, FSH水平異常之間存在劑量效應(yīng)關(guān)系。在調(diào)整可疑混雜因素后，尿液BPA濃度與AD、FSH水平之間存在負相關(guān)關(guān)系，與SHBG、PRL、E2水平之間存在正相關(guān)關(guān)系。 3．尿液中BPA濃度升高與男性性欲沖動程度、受刺激勃起能力、完成性交能力、射精強度、性生活滿意度降低，以及勃起困難程度和射精困難程度升高之間存在劑量效應(yīng)關(guān)系；在調(diào)整可疑混雜因素后，尿液中BPA濃度與各表現(xiàn)指標之間存在相關(guān)關(guān)系。 動物實驗 1．各染毒組體重增長量和染毒后體重之間存在差異，高劑量組體重增長量顯著低于對照組；各染毒組大鼠精囊、前列腺、睪丸、附睪等性腺器官的臟器系數(shù)存在顯著性差異，與對照組相比，中、高劑量組睪丸、精囊腺、前列腺臟器系數(shù)明顯降低，高劑量組附睪臟器系數(shù)也明顯降低。 2．各染毒組間精子質(zhì)量存在顯著性差異；與對照組相比，中、高劑量組精子活力和存活率顯著降低；高劑量組精子密度也顯著降低。 3．各染毒組間性激素T和FSH水平存在顯著性差異；與對照組相比BPA中、高劑量組血清T水平明顯降低，而高劑量組FSH水平明顯升高；各染毒組間AR、FSHR mRNA和蛋白表達平均存在顯著性差異；BPA中、高劑量組大鼠睪丸組織中AR、FSHR mRNA和蛋白水平均顯著低于對照組。 4．未發(fā)現(xiàn)各染毒組間Src mRNA和蛋白以及蛋白磷酸化表達之間存在顯著性差異；未發(fā)現(xiàn)各染毒組間ERK1/2mRNA和蛋白表達之間存在顯著性差異,但ERK1/2蛋白磷酸化表達存在顯著性差異，BPA中、高劑量組ERK1/2蛋白磷酸化水平顯著低于對照組；未發(fā)現(xiàn)各染毒組間CREB mRNA和蛋白表達之間存在顯著性差異,但CREB蛋白磷酸化表達存在顯著性差異，BPA高劑量組CREB蛋白磷酸化水平顯著低于對照組。 結(jié)論 1．尿液BPA水平升高與男性精子質(zhì)量下降、性激素水平異常、性功能下降相關(guān)； 2．高劑量BPA染毒對雄性Wistar大鼠有明顯的生殖毒性，導致性腺器官受損，精子質(zhì)量下降、激素水平改變； 3．BPA可導致大鼠睪丸組織中ERK1/2、CREB蛋白磷酸化水平降低，提示BPA可能通過對ERK1/2、CREB通路的抑制而影響生殖功能，但未發(fā)現(xiàn)Src與這一通路的上游調(diào)控有關(guān)。
[Abstract]:Environmental oestrogen double phenol A (BPA) is an important chemical raw material, and its wide application range leads to ubiquitous exposure. BPA has a weak estrogen-like action and a strong anti-androgen-like action, which can have an impact on the male reproductive system. However, there are very limited reports on population studies and related mechanism studies on BPA male reproductive toxicity. This study explored the reproductive toxicity of BPA to men through population studies, and further explored possible mechanisms through animal experiments to provide scientific basis for determining the male reproductive toxicity and its mechanism of BPA. Objective To explore the effect of BPA exposure on male reproductive function and explore possible mechanisms to study the role of BPA on reproductive function and its mechanism. To explore BPA's relationship between male sperm quality, serum sex hormone level and sexual function by analyzing BPA Reproductive toxicity to men; 2. Study BPA on male rats Effects of colonizing system and sperm quality, as well as on main hormone testosterone (T) and follicle The effect of BPA on hormone receptor AR, FSHR and signal pathway related genes Src, ERK1/ 2 and CREB was studied. Spermatozoa Possible molecular mechanisms in the course of the genesis: Study of population of 543 subjects, general demographic characteristics of subjects using structured questionnaires, past medical history, and use Drug history, lifestyle, history of sexual life, history of BPA exposure, etc. Collection of study subjects urine, semen, blood, application of high performance liquid chromatography (HPLC) to detect BPA levels in urine, computer-assisted sperm analysis system to detect sperm count, activity Serum follicle stimulating hormone (FSH), prolactin (PRL), testosterone (T), estradiol (E2) and enzyme linked immunosorbent assay (ELISA) were detected by radioimmunoassay (RIA). Serum Inhibin B (INB), Free testosterone (FT), Sex Hormone Binding The levels of globulin (SHBG) and androgradeone (AD) were established. The database was established with SPSS10.0 software and t test was used. Methods: Twenty-eight male Wistar rats (2000.20g) were randomly divided into 4 groups according to their weight, the daily fixed time was treated with daily dose of BPA0, 50, 100 and 200mg/ (kg 路 d). Detection of sperm density, viability, survival rate and improved pasteurization in rats by computer-assisted analysis of rat sperm density, viability, survival rate, and epididymal sperm. Serum T and FSH levels were measured by radioimmunoassay. Real-time PCR and Western-blot were used to detect AR, FSHR, Src, E in the testis of rats. RK1/ 2, CREB gene mRNA and protein expression level. Using SPSS10.0 to establish a database and use a single factor variance analysis Statistical analysis was conducted on Dunnet-t test. Results: 1. The increase of BPA concentration in urine was related to low sperm density, low sperm count, low sperm motility, and low sperm survival rate.; after adjusting the suspected confounding factors, urine There was a negative correlation between BPA concentration in liquid and sperm density, total number, living power and survival rate. There was a relationship between elevation and FT, AD, SHBG, PRL and FSH levels. After adjusting the suspected confounding factors, the urine BPA concentration was associated with AD, FSH. There is a negative correlation between the levels, and positive correlation exists between the levels of SHBG, PRL and E2. 3. The increase of BPA concentration in urine and the degree of male sexual desire, stimulated erection ability, completed sexual intercourse ability, ejaculation intensity, sex life satisfaction decreased, and there is a dose between the degree of erection difficulty and the degree of difficulty in ejaculation. effect relationship There was a correlation between BPA concentration in urine and each expression index after adjustment of suspicious confounding factors. Animal experiment 1. There was a difference between body weight growth amount and body weight after exposure, and the growth of body weight in high-dose group was significantly lower than that of control group. There was a significant difference in the organ coefficients of gonadal organs such as seminal vesicle, prostate, testis, epididymis and so on in each group. There was a significant decrease in the organ coefficient of the epididymis in the high-dose group compared with that in the middle and high-dose groups. The sperm quality among the groups was significantly lower than that in the high-dose group. Compared with the control group, the sperm motility and survival rate of the high-dose group decreased significantly; the sperm density of the high-dose group was also significantly decreased. Compared with the control group, the serum T level of the high-dose group was significantly lower than that in the control group. The levels of FSH in the high-dose group were significantly increased, and the expressions of AR, FSHR mRNA and protein in each group There was a significant difference between the levels of AR, FSHR mRNA and protein in the testis tissues of the rats with high dose group. The levels of AR, FSHR mRNA and protein in the testis tissues of the high dose group were significantly lower than those in the control group. There was a significant difference between ERK1/ 2mRNA and protein expression in the group, but there was a significant difference in the phosphorylation of ERK1/ 2 protein. In BPA, the phosphorylation level of ERK1/ 2 protein in high dose group was significantly lower than that in control group. exist Significant differences were noted, but there was a significant difference in the phosphorylation of CREB protein, and the BPA high dose group CREB The level of protein phosphorylation was significantly lower than that in control group. Conclusion 1. The level of BPA in urine and the quality of male sperm decreased, sex hormone level was abnormal, sex hormone level was abnormal, 2. High dose BPA exposure has obvious reproductive toxicity to male Wistar rats, resulting in impaired gonadal organs, decreased sperm quality, changes in hormone levels; 3. BPA can lead to ERK1/ 2, CR in rat testis tissues.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R114

【參考文獻】

相關(guān)期刊論文 前10條

1 張潔;王鴻程;;Src及其抑制劑對非小細胞肺癌作用的研究進展[J];國際內(nèi)科學雜志;2009年01期

2 張玉敏,崔金山,段志文,李海山;環(huán)境雌激素雙酚A對小鼠生精功能的影響[J];工業(yè)衛(wèi)生與職業(yè)病;2004年05期

3 王玉萍;石玉琴;李好文;劉長江;楊克敵;;p,p’-DDE和β-BHC對大鼠支持細胞卵泡刺激素受體的影響[J];環(huán)境與健康雜志;2011年02期

4 張新宇;李漢忠;李宏軍;;雄激素受體基因敲除對精子發(fā)生影響的研究進展[J];基礎(chǔ)醫(yī)學與臨床;2010年09期

5 陳浩宇;王水明;彭瑞云;董霽;左紅艷;王麗峰;高亞兵;徐新萍;蘇鎮(zhèn)濤;;微波輻射對大鼠睪丸組織pCREB、CREM及CBP蛋白表達的影響[J];解放軍醫(yī)學雜志;2012年04期

6 陳雪雁,陳實平,金淑敏,程凱,董紅燕,陳克銓;雄激素受體和卵泡刺激素受體在成年大鼠睪丸中的期依賴性表達[J];解剖學報;2001年04期

7 石峻嶺,楊水蓮,肖國兵,鄭力行,周志俊;普通人群血清雙酚A水平的測定[J];環(huán)境與職業(yè)醫(yī)學;2004年03期

8 肖國兵,石峻嶺,何國華,鄭力行,吳同俊,周志俊;環(huán)氧樹脂生產(chǎn)工人血清雙酚A與性激素水平的調(diào)查[J];環(huán)境與職業(yè)醫(yī)學;2005年04期

9 田二坡;秦達念;;雄激素受體在睪丸內(nèi)的分布及與生精調(diào)節(jié)的關(guān)系[J];中國男科學雜志;2007年05期

10 杜鵑;胡淼;崔克勤;;雙酚A對小鼠生殖內(nèi)分泌影響的實驗研究[J];中國熱帶醫(yī)學;2007年06期

相關(guān)博士學位論文 前2條

1 馬艷華;嚴重少精子癥患者血清相關(guān)激素測定與精子超微形態(tài)及Y染色體微缺失檢測研究[D];天津醫(yī)科大學;2007年

2 陳雪梅;鄰苯二甲酸二丁酯和苯并[a]芘對雄性大鼠的聯(lián)合生殖毒性研究[D];重慶醫(yī)科大學;2009年

相關(guān)碩士學位論文 前1條

1 關(guān)霞;p，p'-DDE、β-BHC聯(lián)合作用對大鼠睪丸支持細胞Ca~（2+）、ERK和BCL-2表達的影響[D];華中科技大學;2009年

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