【作者】 常玲玲；

【导师】 佘锐萍；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2014， 博士

【摘要】 2008年中国婴幼儿因食用非法掺杂三聚氰胺(melamine, MA)的配方奶粉爆发尿结石,该事件引起了世人对三聚氰胺或与其同系物三聚氰酸(cyanuric acid, CA)协同作用CMA+CA, MC)对人和动物健康影响的广泛关注。过去大量的研究都集中在MA及其同系物对泌尿系统的毒性作用上,对其他系统的毒性作用研究报道还很少见。本研究采用组织细胞病理学和超微病理学观察技术、免疫组化、Western Blot技术、细胞培养和流式细胞术等方法,在原有研究的基础上,以血睾屏障和支持细胞为切入点,从毒理病理学角度观察研究了MA及其同系物对小鼠生殖系统的毒性作用,在整体水平、组织学水平、细胞学水平、超微水平和分子水平上多角度、系统地揭示了MA对小鼠生殖系统的毒性作用及其机理。研究结果如下：1.体内试验以8周龄雄性昆明小鼠为试验对象,设立对照组,MA组和MC组,其中MA组和MC组分别设立低、中、高三个剂量组,MA剂量分别是30、140和7OOmg/kg.bw/d, MC剂量分别是15(15MA+15CA).70(70MA+70CA)和350(350MA+350CA) mg/kg.bw/d, MC由相同剂量的MA和CA混合。采用灌胃的方式染毒,连续3天,染毒后第1天(Post1d)和第5天(Post5d)剖检取材,评价MA和MC的雄性生殖毒性效应,观察血睾屏障结构的变化,探讨它们对睾丸毒性作用的靶细胞。通过临床观察、血清学分析、剖检及组织病理学观察和精子指标检测可见,Post1d时,与对照组相比,MC各剂量组体重增长均显著下降(P<0.05或P<0.01),中、高剂量组小鼠出现明显的临床症状;高剂量组睾酮水平显著下降(P<0.01),睾丸和附睾脏器指数显著下降(P<0.05或P<0.01)。所有MA和MC组小鼠的睾丸和附睾组织出现了明显的病理学损伤,呈现剂量相关性,病变主要表现为生精细胞脱落和生精小管上皮空泡化,附睾输出管中充满多量脱落的生精上皮细胞,严重者出现少精或无精；所有处理组精子畸形率均显著升高(P<0.05或P<0.01)。到Post5d时,所有处理组精子畸形率仍然是显著升高(P<0.05或P<0.01),其它检测指标显示睾丸毒性程度减轻。以上结果表明,MA和MC对睾丸生精上皮具有直接的损伤作用,从而导致精子质量下降。利用TUNEL染色观察睾丸细胞凋亡情况,并用免疫组化和Western Blot定位和定量检测了Fas/Fas-L蛋白在睾丸组织中的表达变化。结果可见：Postld时,与对照组相比,处理组睾丸生精细胞出现明显凋亡,MA低剂量组和MC各剂量组睾丸凋亡指数显著升高(P<0.05或P<0.01),Fas和Fas-L分别在生精细胞和支持细胞中有不同程度的表达；Post5d时,与对照组相比,MA高剂量组和MC中、高剂量组凋亡指数仍显著升高(P<0.05或P<0.01),MA与MC高剂量组Fas和Fas-L均在生精细胞中出现强阳性表达。上述结果提示,MA和MC可以促进小鼠睾丸的生精细胞凋亡。利用透射电镜观察血睾屏障超微结构变化,并用免疫组化和Western Blot定位和定量检测了各层血睾屏障结构中关键蛋白的表达变化。结果发现：Postld时,与对照组相比,所有MA与MC组的小鼠血睾屏障各层超微结构出现了明显的损伤,主要表现有：毛细血管内皮细胞、肌样细胞和支持细胞胞浆中出现大量空泡,支持细胞间的紧密连接出现明显裂隙；支持细胞中的波形蛋白出现崩解,表达量明显下降(P<0.05或P<0.01),睾丸组织中β-actin蛋白表达量不同程度出现显著下降(P<0.05或P<0.01)。Post5d,各处理组血睾屏障的超微结构损伤仍很严重,波形蛋白表达量仍下降(P<0.05或P<0.01)。以上结果表明,MA和MC可以破坏小鼠睾丸血睾屏障结构,导致支持细胞骨架结构崩解,说明血睾屏障结构的破坏在MA和MC引发的雄性生殖毒性作用中发挥了关键的作用。2.体外试验以小鼠TM4支持细胞株为试验对象,采用MTT、透射电镜、流式细胞仪、Western Blot等方法,观察不同浓度MA(0-1000ug/mL)对支持细胞的毒性作用。结果可见,TM4细胞在50ug/mL及以上的MA作用24h后,细胞存活率显著下降(P<0.05或P<0.01)；在30-500ug/mL的MA作用24h后,细胞的超微结构出现了明显损伤,高浓度MA促进细胞凋亡(P<0.05或P<0.01)；在50ug/mL MA分别作用0、1、3、6、24h后,波形蛋白随着时问延长表达量下降。以上结果表明低浓度的MA即可对TM4支持细胞产生明显的损伤,提示支持细胞可能是MA生殖毒性作用的重要靶点细胞。上述体内和体外试验观察研究结果表明,在本试验剂量条件下MA单独和MC混合灌服均可引起雄性小鼠睾丸组织明显的损伤,精子畸形率升高,造成损伤的主要机制可能是由于MA和MC靶向于支持细胞破坏血睾屏障结构,继而引起各级生精细胞发生变性坏死、凋亡加剧,以致精子生成障碍、发育畸形。更多还原

【Abstract】 In2008in China, the occurrence of an outbreak of urinary stones in infants draw critical attention to the toxicity of melamine (MA) or the mixture of MA and cyanuric acid (CA) in human and animals.Until now, a large number of researches focused on the toxic effects of MA and its analoges on the urinary organs. In this study, based on our prelimitary results, we focused on the blood-testis barrier and Sertoli cells to investigate the toxic effects of MA and its analogues on male reproductive system on different levels with methods of histochemical and ultrastructural pathology,immunohistochemical staining, Western blot and flow cytometry. The research contents are as follows:1. In vivo studyEight-week-old male Kunming mice were administered either melamine (MA,30,140, or700mg/kg/day), a melamine and cyanuric acid mixture (MC, each at15,70, or350mg/kg/day), or vehicle (control) for3consecutive days. Testicular toxicity was evaluated on days1(Post Id) and5(Post5d) after the final exposure. Furthermore, the structure of blood-testis barrier and possible cellular target for MA and MC was investigated.With the methods of clinical observation, serum chemistry analysis、autopsy、histopathological examination and sperm detection, on Post1d, we found that the body weight gain of the MC-treated mice significantly decreased (P<0.05or P<0.01), with obvious clinical sighs in the higher groups. In the highest MC group, the serum T level significantly decreased(P<0.01), and the organ indexes of testis and epididymis decreased (P<0.05or P<0.01). In both MA and MC groups, the testes and epididymis tissue showed dose-related histopathological lesions, and the characterize changes were germ cell exfoliation and tubular vacuolation. Additionally, the sperm abnormality were significantly increased in all treated groups (P<0.05or P<0.01). On Post5d, the sperm abnormality were still significantly increased in all treated groups (P<0.05or P<0.01).The other indexes indicated that the degree of testicular injury became slighter at this time point. These results suggested that MA and MC had direct toxicity on the testicular epithelium and caused decrease of sperm quality.We detected the apoptosis and the protein expression of Fas/Fas-L in the testes with TUNEL staining, immunohistochemistry staining and Western Blot. On post Id, the results showed that the germ cells exhibited apoptosis in the testes among all treated groups, and apoptotic index was significantly increased in low MA group and all MC groups(P<0.05or P<0.01). The Fas and Fas-L distributed in the germ cells and Sertoli cells, respectively. On post5d, apoptotic index in the highest MA group and the highest two MC groups(P<0.05or P<0.01). In the highest MC group, the Fas and Fas-L protein showed strong expression in germ cells. These results suggested that MA and MC could induce germ cell apoptosis, and the Fas/Fas-L system may involved the apoptotic process.The structure of blood-testis barrier and related protein was evaluated with TEM, immunohistochemistry staining and Western Blot. On Post Id, the results showed that structure of the blood-testis barrier was damaged dose dependently. The vimentin filament in Sertoli cell collapsed in all treated groups. The expression of vimentin and β-actin protein decreased with difference degree among the treated groups (P<0.05or P<0.01). On Post5d, the lesions of the blood-testis barrier structure was still serious in the treated groups, with decreased expression of vimentin protein(P<0.05or P<0.01). These results suggested that MA and MC could disrupt the blood-testis barrier and decreased cytoskeleton protein expression. Sertoli cells appear to be a cellular target for MA and MC.2. In vitro studyWith the methods of MTT, TEM, Flow cytometry and Western Blot, we evaluated the cytotoxicity of MA (0-1000ug/mL) on TM4Sertoli cells. When cells were treated with MA at50ug/mL or above for24h, the cell viability was significantly decreased(P<0.05or P<0.01). When cells were treated with MA at range of30to500ug/mL for24h, the ultrastructure was obviously damaged, and high concentration of MA induced significant apoptosis(P<0.05or P<0.01). When cells were treated with MA at50ug/mL MA for0,1,3,6or24h, the expression of vimentin protein decreased with time. It can be seen that MA even at low concentration could induce obvious injury in TM4cells, suggesting that Sertoli cells maybe sensitive to MA toxicity.Taken together, the in vivo and in vitro studies showed that oral exposure to MA and MC in male Kumming mice could produce obvious testicular toxicity including germ cell sloughing and apoptosis and high rate of sperm abnormality. MC exhibited much more toxicity than MA. The damage of blood testis barrier and Sertoli cell play a critical role in the MA-and MC-induced male reproductive toxicity.更多还原