【作者】 耿江；

【导师】 丁强； 陈刚； 吴忠； 方祖军；

【作者基本信息】 复旦大学 ， 泌尿外科， 2009， 博士

【摘要】 第一部分丙烯腈对男性接触人群精子质量的影响目的探讨丙烯腈（AN）对男性接触人群精子质量及血清激素水平的影响。材料与方法采集94例男性精液标本,其中健康志愿者27例作为对照组,丙烯腈接触工人67例作为接触组,采用计算机辅助精子分析技术（CASA）分析其精子质量。改良巴氏染色观察精子畸形率。采用电化学发光免疫法检测血清中促甲状腺激素（TSH）,卵泡刺激素（FSH）,黄体生成素（LH）,雌激素（E₂）及睾酮（T）的水平。单细胞凝胶电泳法检测精子DNA完整性。结果丙烯腈接触组男性的精子密度、运动精子密度、前向运动精子密度、精子运动速度（VAP）、精子前向运动速度（VSL）以及直线性（LIN）和摆动性（WOB）均较对照组降低,精子畸形率及液化时间较对照组升高。丙烯腈接触大于15年的男性工人的运动精子密度、前向运动精子密度、精子运动速度、精子前向运动速度、直线性及摆动性低于丙烯腈接触小于等于15年的男性和对照组男性。接触组和对照组的促甲状腺激素、黄体生成素、卵泡刺激素无明显差异,接触组雌激素水平高于对照组,差异有统计学意义,而睾酮虽然低于对照组,但无统计学意义。接触组精子细胞的尾长、尾部DNA含量、Olive尾矩均大于对照组,但仅尾部DNA含量与对照组相比有统计学意义。结论丙烯腈接触能导致精子质量特别是精子运动参数的降低及性激素水平的紊乱。应用计算机辅助精子分析技术能够客观、准确地评估丙烯腈接触对男性精子的影响。第二部分丙烯腈致睾丸毒性的实验研究目的探讨丙烯腈（AN）对大鼠精子质量的影响及其致睾丸毒性的分子机制。材料与方法采用含不同浓度AN的蒸馏水对SD大鼠进行灌胃,浓度分别为0,5,10,20 mg/kg/d,共10周。每周测量大鼠体重。10周后,处死大鼠,获取大鼠睾丸、附睾,行组织病理学检查。电镜观察睾丸超微结构变化。采用计算机辅助精子分析技术检测大鼠附睾精子质量。通过检测大鼠睾丸还原型谷胱甘肽（GSH）和丙二醛（MDA）含量,谷胱甘肽过氧化物酶（GSH-Px）和超氧化物歧化酶（SOD）的酶活性,评估睾丸的氧化应激情况。酶联免疫法检测大鼠血清睾酮水平。免疫组织化学检测睾丸组织中Fas、FasL的表达。RT-PCR和Western-Blot检测睾丸组织Fas,FasL和3β羟基类固醇脱氢酶（3β-HSD）mRNA和蛋白质表达水平。结果随着AN灌注浓度的升高,大鼠体重呈下降趋势,但睾丸附睾重量无明显变化。实验组大鼠的附睾精子活率、精子前向活动率、VAP、VSL、LIN明显降低,并呈剂量依赖关系。电镜下发现实验组大鼠睾丸的组织病理学改变主要为以支持细胞空泡化及Leydig细胞凋亡为特征。免疫组织化学检测发现Fas表达于睾丸间质Leydig细胞、FasL表达于睾丸支持细胞（Sertoli）和间质细胞（Leydig）,且随着AN灌注浓度的升高,Fas/FasL表达呈增高趋势。实验组大鼠睾丸GSH含量显著降低,而MDA含量显著升高。实验组大鼠睾丸Fas和FasL mRNA和蛋白质表达水平显著升高,而3β-HSD表达水平显著降低。结论丙烯腈接触对大鼠精子运动能力有抑制作用。氧化应激损伤在丙烯腈所致的睾丸毒性中起重要作用,其机制可能涉及Fas/FasL系统的参与及睾丸Leydig细胞的凋亡。第三部分抗氧化剂genistein对丙烯腈致大鼠Leydig细胞毒性的拮抗效应目的探讨抗氧化剂genistein对丙烯腈（AN）致大鼠Leydig细胞毒性的拮抗效应。材料与方法采用酶消化法结合Percoll非连续密度梯度法分离纯化大鼠睾丸Leydig细胞,并行3β-HSD特异性免疫荧光染色鉴定。CCK8法检测不同浓度AN对Leydig细胞的毒性作用。RT-PCR和Western blot检测AN对Leydig细胞Fas/FasL表达的影响。流式细胞术分析AN以及抗氧化剂genistein对Leydig细胞凋亡的影响。测定genistein对AN处理的Leydig细胞MDA及GSH含量的影响,并检测genistein对AN处理的Leydig细胞NFκB核蛋白表达的影响。结果酶消化法结合Percoll非连续密度梯度法能获得较高纯度的大鼠Leydig细胞。AN对睾丸Leydig细胞的细胞毒性具有时间剂量依赖关系。流式细胞术检测发现Leydig细胞凋亡显著增加。AN处理的Leydig细胞Fas、FasL表达明显升高,MDA含量增加,GSH含量减少。AN和genistein共处理的Leydig细胞凋亡明显减少,MDA含量降低,GSH含量升高,NF-κB p65蛋白表达增高。结论丙烯腈能通过诱导大鼠Leydig细胞氧化应激和Fas/FasL表达而促进细胞凋亡。抗氧化剂genistein可能通过NF-κB信号转导通路抑制丙烯腈所致的Leydig细胞氧化应激损伤和凋亡。更多还原

【Abstract】 PartⅠEffect of acrylonitrile exposure on semen quality among occupational workersAim To investigate the effects of acrylonitrile exposure on the semen quality of occupational workers.Materials and methods Sixty-seven male workers who were exposure to acrylonitrile and 27 male healthy volunteers in the same area were selected as the exposure group and control group,respectively.The men provided information relating to lifestyle,occupational details.After the semen was collected according to the standard method,the workers’ semen qualities were analyzed by the WLJY-9000 CASA（computer-assisted semen analysis） system.Serum concentrations of thyrotropic stimulating hormone（TSH）、follicle stimulating hormone（FSH）、luteotrophic hormone（LH）、testosterone（T） and estradiol（E₂） were analyzed by electro-chemiluminescence immunoassay.Sperm DNA integrity was assessed using the single-cell gel electrophoresis（SCGE） assays.Results Significant differences were observed in the sperm concentration,the concentration of motile and progressive sperm,straight line velocity（VSL）,average path velocity（VAP）,linearity（LIN） and wobble coefficient（WOB）.Through the analysis of multiple regression,we obtained age and exposure time（seniority） showed a significant relationship with the sperm concentration,the concentration of progressive sperm,and average path velocity （VAP）.In exposure group,the levels of E₂ increased significantly and T decreased, although there was no significant decrease in the T levels between two groups. Statistically significant increasing was found in sperm DNA damage in exposure group compared to control group for the tail moment,otherwise tail length and Olive tail moment have no significant increasing.Conclusions Occupational exposure to acrylonitrile could affect the semen quality and sexual hormone levels of the workers, especially,the sperm movement parameters.This appears to be the first study showing the value of CASA in observation of reproductive toxicity of acrylonitrile exposure in the male workers.The application of CASA provides a plausible and objective method to predict the fertility of male AN-exposed workers. PartⅡExperimental study of testicular toxicity induced by acrylonitrileAim To investigate the acrylonitrile induced testicular toxicity and oxidative stress and associated Fas/Fas ligand（FasL） expression.Material and methods Sprague-Dawley rats were administer drinking water by gavage formulated to contain 0,10,15,20 mg per kg body weight per day for ten weeks,respectively.Rats were closely monitored clinically with body weight every week.After ten weeks,all rats were necropsied and then genital organ weights and histopathology were examined. Ultrastructural studies of testes were observed under transmission electron microscope. The epididymal sperm motile parameters were analyzed by computer-assisted semen analysis system.Oxidative stress was assessed in testes by determining glutathione （GSH） level,glutathione peroxidase（GSH-Px） activity,superoxide dismutase（SOD） activity and lipid peroxidation as indicated by malondialdehyde（MDA） production. Serum testosterone level was assessed by enzyme linked immunosorbent assay （ELISA）.Fas and FasL expression in rat testis were examined by immunohistochemistry.Expression levels of Fas,FasL and 3β-hydroxysteroid dehydrogenase（3β-HSD） were analyzed by RT-PCR and Western blotting.Results Our data showed significant changes in the weight of the body,however,no obvious differences in the weight of testis and epididymis were found after AN treatment. Significant reducing was observed in the parameters of the percentage of motile sperm,the percentage of progressive motile sperm,VAP,VSL and LIN in a dose dependent manner.Histopathologic evaluations demonstrated that massive vacuolization appeared in the seminiferous epithelium and Leydig cells apoptosis were found in interstitial tissue of AN treated rat testis which consistent with the decreasing serum testosterone level in AN treated rat.Immunohistochemistry experiments in AN treated rat testis demonstrated the increasing expression of FasL in Sertoli and Leydig cells whereas the increasing expression of Fas was confined to the Leydig cells.AN reated testis revealed a significant decrease in testicular GSH content as well as a significant enhancement of MDA accumulation in a dose-dependent manner.We found a significant increase in expression of Fas and FasL mRNA and protein in AN treated rat testis.Moreover,there was a significant decrease in the levels of 3beta-HSD mRNA and protein of AN treated testis.Conclusions Our results suggest that AN exposure had side effect on sperm mobility of rats.Oxidative stress may play a role in the testicular toxicity induced by AN,and the Fas/FasL system may be involved in the oxidative damage and the apoptosis of Leydig cells.PartⅢGenistein prevents acrylonitrile-induced toxicity in primary cultured rat Leydig cellsAim To determine the effect of AN on the viability of primary-cultured rat Leydig cells as well as the oxidative damage generated by AN by measuring MDA and GSH levels in primary cultured Leydig cells.We also analyzed whether the AN toxicity could be avoided by using antioxidant genistein.Material and methods Leydig cells were separated by enzymatic digestion combined with discontinuous Percoll density gradient centrifugation.Cells were exposed in vitro to different concentrations of AN for different time intervals.Cell toxicity was measured by CCK8 assay.Oxidative stress was assessed by determining glutathione（GSH） level and malondialdehyde （MDA） production.Expression levels of Fas and FasL were assessed by RT-PCR and Western blotting.Apoptosis rate was analyzed by Annexin V-FITC/PI flow cytometry （FCM） and the expression of NF-κB p65 was measured by Western blot.Results Our data show that AN inhibited the growth in a time and dose dependent manner.The expression of Fas and FasL was significantly elevated in a time dependent manner. AN enhanced oxidative stress,as indicated by malondialdehyde（MDA） accumulation, and depleted reduced glutathione（GSH） level.Leydig cell exposed to AN treated with genistein afford some protection,as indicated by reducing MDA level and elevating level of GSH.Furthermore,co-treatment with genistein and AN cause a decrease in apoptosis rate of Leydig cells.Meanwhile,the expression of NF-κB p65 increased significantly.Conclusions Our study indicated the apoptotic effect of acrylonitrile on Leydig cells might be through oxidative stress and Fas/FasL system.Genistein could prevent acrylonitrile-induced oxidative stress and apoptosis of Leydig cells,which may be involved NF-κB signal transduction pathways.更多还原