双酚A暴露对子鼠雄性生殖细胞凋亡的影响

发布时间：2018-06-01 13:51

本文选题：双酚A + 睾丸生殖细胞　；参考：《安徽医科大学》2013年硕士论文

【摘要】：背景：双酚A(bisphenol A, BPA)是环境雌激素中最常见的一种,属于全球性的环境污染物,随着科技的发展而应用到日常生活中的很多领域。研究表明BPA的半衰期较长,对机体的生殖系统、免疫系统、神经系统和胚胎发育等方面都有不利的影响,甚至可使机体发生癌变等,严重威胁着人类及后代的健康。已有实验表明BPA对机体的生殖功能产生不可逆的破坏作用,特别是对雄性生殖系统的发育。然而孕期BPA暴露对子代雄性生殖毒性仍然有不同的看法。目的：本研究观察BPA暴露对子鼠雄性生殖细胞的形态学变化和细胞凋亡的影响,进一步探讨BPA对雄性生殖系统的影响及可能机制。方法：ICR孕鼠随机分为空白对照组、溶剂对照组、BPA低剂量组(10nmol/L)、BPA中剂量组(100nmol/L)和BPA高剂量组(1000nmol/L),每组8只；孕鼠饮水染毒至小鼠断乳后42d[出生后天数(postnatal day, PND42)],于PND21和PND42分别处死雄性子鼠。进行以下研究：(1)统计每组每只孕鼠生产子鼠的数目及雌雄比例；计算睾丸脏器系数(睾丸湿重与子鼠体重比值)；(2)H.E染色观察子鼠睾丸组织的形态学变化；(3)透射电镜观察睾丸组织电镜改变；(4)Hoechst33258染色荧光显微镜检测观察子鼠睾丸生殖细胞凋亡情况；(5)免疫组化染色SABC法检测子鼠睾丸生殖细胞Caspase-3的表达情况；(6)Western blot检测子鼠睾丸组织匀浆Caspase-3的表达量。结果：各组每只母鼠的子鼠数量和雌雄比例与对照组相比差异无显著性(P0.05)。随着BPA染毒剂量的增加,PND21和PND42的实验组小鼠的睾丸脏器系数呈下降趋势,以中、高剂量组更为明显。H.E染色显示,BPA暴露组子鼠睾丸生精小管管壁细胞层数减少,细胞排列零乱,其中6W的中、高剂量组较为明显；电镜结果显示BPA中、高剂量组小鼠睾丸内精原细胞、支持细胞和间质细胞呈现病理学改变,精原细胞、支持细胞和间质细胞内的线粒体嵴消失呈明显空泡样改变,双层膜结构不清,粗面内质网出现不同程度的扩张,结构零乱松散,且有些细胞线粒体数目减少,细胞核染色质边集；Hoechst33258染色结果示：中剂量组精原细胞、支持细胞和间质细胞的核呈致密浓染,使细胞核呈现高强度的固缩状态的蓝色荧光,即细胞发生凋亡；Western blot和免疫组化结果均显示：BPA中、高剂量组子鼠睾丸生殖细胞Caspase-3的表达增加,且多位于睾丸间质细胞中和靠近基膜的精原细胞和支持细胞中。结论：出生前后暴露BPA可使小鼠的睾丸脏器系数下降,生精细胞、支持细胞和间质细胞发生形态学改变和细胞凋亡增加等,说明BPA可能通过胎盘屏障和母乳影响雄性子鼠生殖系统的发育,其机制可能与增加雄性子鼠生殖细胞的凋亡有关。
[Abstract]:Background: bisphenol A (BPAA) is one of the most common environmental estrogens, which is a global environmental pollutant. With the development of science and technology, it has been applied in many fields of daily life. The results show that BPA has a long half-life, which has adverse effects on the reproductive system, immune system, nervous system and embryonic development of the body, and even makes the body cancerous, which seriously threatens the health of human beings and their offspring. It has been shown that BPA has irreversibly destructive effects on reproductive function, especially on the development of male reproductive system. However, BPA exposure during pregnancy still has different views on male reproductive toxicity in offspring. Aim: to observe the effects of BPA exposure on the morphological changes and apoptosis of male germ cells in rats and to explore the effects of BPA on the male reproductive system and its possible mechanism. Methods Twenty ICR pregnant mice were randomly divided into two groups: control group (n = 10 nmol / L) and high dose BPA group (n = 8). The pregnant mice were exposed to drinking water until 42 days after weaning (postnatal day (PND 42), and the male mice were killed at PND21 and PND42, respectively. Do the following research: 1) count the number and the ratio of male and female to each group of pregnant rats; Calculation of testicular visceral coefficient (ratio of wet weight of testis to body weight of the offspring) the morphological changes of testis were observed by means of electron microscopy (transmission electron microscope (TEM) observation of testicular tissue changes by fluorescence microscopy with Hoechst33258 staining The expression of Caspase-3 in testicular germ cells was detected by immunohistochemical SABC method. The expression of Caspase-3 in testis homogenate was detected by Western blot. Results: there was no significant difference in the number and ratio of female and male between each group and the control group (P 0.05). With the increase of the dose of BPA, the testicular viscera coefficient of mice in PND21 and PND42 groups showed a decreasing trend. In the middle and high dose groups, the number of testicular seminiferous tubule wall cells decreased and the cells arranged in disorder in the middle and high dose groups. The results of electron microscope showed that the spermatogonium, Sertoli cells and interstitial cells in the testis of BPA mice showed pathological changes. In Sertoli cells and interstitial cells, the mitochondrial cristae disappeared with obvious vacuolar changes, the double layer membrane structure was unclear, the rough endoplasmic reticulum expanded to varying degrees, the structure was loose, and the number of mitochondria in some cells decreased. The results of Hoechst33258 staining showed that the nuclei of Sertoli cells and interstitial cells in the middle dose group were dense and dense, resulting in a high intensity blue fluorescence of the nucleus in the pyknotic state. The results of Western blot and immunohistochemistry showed that the expression of Caspase-3 in testis germ cells increased in high dose group, and most of them were located in stromal cells of testis and spermatogonia and Sertoli cells near the basement membrane. Conclusion: exposure to BPA before and after birth can decrease the testicular organ coefficient and increase the morphological changes and apoptosis of spermatogenic cells Sertoli cells and interstitial cells in mice. It is suggested that BPA may influence the development of reproductive system of male offspring through placental barrier and breast milk, and its mechanism may be related to increasing the apoptosis of germ cells in male offspring.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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