【作者】 姜春明；

【导师】 孙志伟；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2004， 硕士

【摘要】 甲基汞是严重威胁生态环境和人类健康的一类重要污染物。环境中的甲基汞可以通过生物富集作用对机体产生毒作用，引起甲基汞中毒。有研究表明，甲基汞可以诱发体细胞及生殖细胞DNA损伤，损害雄性生殖系统和雌性生殖系统，进而影响子代的生长发育。我们在研究中发现：1、甲基汞对体外睾丸生殖细胞DNA的损伤效应应用单细胞凝胶电泳的方法检测甲基汞对睾丸生殖细胞DNA链的损伤，结果表明0.0001mmol/L的甲基汞即可引起生殖细胞DNA损伤，随着甲基汞剂量的增加，损伤程度逐渐加重。各染毒剂量组与阴性对照组相比较差异具有显著性（P＜0.05），各染毒剂量组间相比较差异无显著性。提示：甲基汞可以造成睾丸生殖细胞DNA不同程度的损伤。2、HE染色观察甲基汞对睾丸生殖细胞凋亡的影响 经苏木精—伊红染色，光学显微镜下观察，甲基汞染毒组睾丸组织中均有散在的凋亡细胞。细胞胞浆深染，呈淡红色，细胞核固缩，呈蓝黑色。提示：甲基汞可以导致睾丸生殖细胞凋亡。3、TUNEL法观察甲基汞对睾丸生殖细胞凋亡的影响TUNEL染色是一种快速而便捷的凋亡检测手段。实验发现甲基汞染毒组的凋亡细胞散在分布，细胞核或胞浆呈棕黄色或黄色着染。图像分析结果表明1/100LD50染毒组凋亡指数高于对照组凋亡指数，但是无显著性差异。随着染毒剂量的增加，1/50LD50、1/10LD50组与对照组比较有显著性差异。提示：进一步证明甲基汞可以导致睾丸生殖细胞凋亡，并且随着剂量的增加凋亡指数增大。 <WP=51>4、免疫组织化学法观察Fas表达情况在细胞凋亡的过程中，Fas是重要的死亡受体。我们在实验中观察到甲基汞染毒组阳性细胞胞膜或胞浆呈棕黄色、深棕黄色染色。图像分析结果表明1/100LD50染毒组Fas表达高于对照组，随着染毒剂量的加大，各组平均灰度值逐渐增大，Fas表达降低。各染毒组与对照组比较无显著性差异。组间比较：1/100LD50组与1/10LD50组、1/50LD50组与1/100LD50组有显著性差异。5、免疫组织化学法观察FasL表达情况实验观察发现甲基汞染毒组阳性细胞胞膜或胞浆呈深棕黄色染色。图像分析结果表明1/100LD50染毒组FasL表达高于对照组，随着染毒剂量的加大，各组平均灰度值逐渐增大， FasL表达降低。各染毒组与对照组比较无显著性差异。6、免疫组织化学法观察caspase-3表达情况Caspase-3是引起细胞凋亡级联反应的关键酶。实验发现：甲基汞染毒组caspase-3阳性表达为细胞膜或细胞浆呈棕黄色染色。图像分析结果表明1/100LD50组与对照组比较具有显著性差异。随着剂量的增加，平均灰度值逐渐加大，caspase-3表达降低。本文应用卫生毒理学和分子生物学方法，从分子水平研究了甲基汞对雄性小鼠生殖细胞的损伤作用和导致其凋亡的调控过程，实验表明本项研究对于揭示甲基汞诱导睾丸生殖细胞凋亡的调控机制以及甲基汞中毒的机理具有重要的理论意义，为防治甲基汞中毒提供了新的思路。更多还原

【Abstract】 Methylmercury(MeHg) is an important environmental pollutant which seriously threaten the ecological environment and the people’s health. The study has shown that MeHg could induce the DNA damage of somatic cells and germ cells. It has effects on female and male reproductive system and affects the growth of the offspring. We used the techniques of toxicology and molecular biology to study the DNA damage and the apoptosis of germ cells in male mice induced by MeHg. The results showed as follows: 1. The effects of MeHg on DNA damage of germ cells in male mice in vitro DNA dmage was observed in 0.001mmol/L group with SCGE. There was a positive correlationship between the exposure doses of MeHg and rate of DNA migration(P＜0.05). But there were no significant differences among exposed groups. It indicated that MeHg could induce the DNA damage of germ cell to some degree. 2. The effects of MeHg on apoptosis of testis germ cell by HE Dispersed apoptotic cells in testis tissues were observed in all of groups treated with MeHg by hematoxylin-eosin dyeing, the plasm of these cells took on light red, the nuclei shrinked and took on dark blue. These suggested that MeHg could induce apoptosis of testis germ cell. 3. The effects of MeHg on apoptosis of testis germ cell by <WP=53>TUNEL TUNEL is a convenient method to assess apoptosis. We found apoptotic cells dispersed in testis, the nuclei or plasm of them took on brownish-yellow or yellow by staining. The results of image analysis showed that the apoptotic index of the groups treated with MeHg was higher than that in the control group, the apoptotic index was significantly elevated in the 1/50LD50 and 1/10 LD50 MeHg treatment groups compared with that in the control group. All above further confirmed MeHg could induce apoptosis of testis germ cell, and with increasing dose of MeHg, apoptotic index increased gradually.4. The expression of Fas/FasL by immunohistochemical study Fsa/FasL is an important death receptor in the process of cell apoptosis. We found that the membrane or plasm of the cells which expressed positively Fsa/FasL of the groups treated with MeHg took on brownish-yellow or dark brownish-yellow by staining. The results of image analysis showed that the expression of Fsa/FasL wasn’t significant difference compared with that in the control groups. But the expression of the group treated with 1/100LD50 MeHg was more than that in the control groups. With the increasing dose of MeHg, the average value of gray scale of every group increased gradually, which represent that the expression of Fsa/FasL decreased.5. The expression of caspase-3 by immunohistochemical study Caspase-3 is a main enzyme which can trigger cell apoptotic cascade reaction. The results of this study showed that the <WP=54>membrane or plasm of the cells which expressed caspase-3 positively of the groups treated with MeHg took on brownish-yellow by staining. The results of image analysis showed that when treated with 1/100LD50 MeHg, the expression of caspase-3 was significant difference compared with that in the control group. With the increasing dose of MeHg, the average value of gray scale of every group increased gradually. Which represent that the expression of caspase-3 decreased.The above-mentioned results had a very significant effect on revealing the regulatory mechanism of apoptosis of testis germ cell by MeHg, and the mechanism of MeHg poisoning. At the same time, they provided new thinking for prevention and cure of MeHg poisoning.更多还原