【作者】 刁书永；

【导师】 袁慧；

【作者基本信息】 湖南农业大学 ， 临床兽医， 2005， 硕士

【摘要】 镉是常见的环境和工业污染物,对雌性哺乳动物生殖系统具有明显的毒性。可引起卵巢病理组织学改变,干扰排卵和受精过程,引起暂时性不育,还可能对雌激素受体、孕酮受体及基因表达产生影响。 研究目的:探讨镉对卵巢颗粒细胞的凋亡诱导作用,阐明其作用机理;在理论上对镉的性腺毒性作用给予补充,为今后的研究提供理论依据。 研究方法:本实验采用25日龄未成年雌性小鼠为实验对象,通过孕马血清促性腺激素处理后再腹腔注射氯化镉的在体实验模型,采用病理学方法检测卵巢中卵泡闭锁的变化:用DNA ladder的有无及TUNEL法来检测卵巢颗粒细胞是否凋亡及凋亡率的变化;用免疫组化方法检测颗粒细胞bcl-2、bax基因的表达;用亚硒酸钠作为拮抗剂来探讨硒对镉毒性的拮抗作用。 实验结果:用琼脂糖凝胶电泳法未能检测到48h各组的DNA梯状带,96h仅高剂量组(4mg/kg·wb)有梯状带出现。TUNEL法检测结果为:48h对照组凋亡率为0.6378%;高、中、低剂量组分别为13.8871%、10.7573%、10.0208%;硒组为5.7232%。96h对照组凋亡率上升为11.0963%,高、中、低各组分别为20.3204%、12.2031%、12.2171%,硒组为14.9203%。表明各组均有不同程度的凋亡,且随镉剂量增加,凋亡率有上升趋势,差异有极显著性(P<0.01)。病理学检察卵巢切片中卵泡闭锁情况,结果随镉剂量的增加,卵泡闭锁率逐渐升高,差异有显著性。免疫组化法检测Bax和Bcl-2蛋白表达,结果Bax在各组普遍表达,并随镉剂量的加大而表达增强,实验组与对照组间差异极显著(P<0.01),48h与96h两时间段间无差异;Bcl-2则普遍表达较弱或不表达,各组间无显著性差异。 实验结论:1) 镉能促进小鼠卵巢颗粒细胞凋亡,并具有剂量效应关系。2) 镉能促进卵泡闭锁,使卵巢间质腺数量增加。3) 镉能增强颗粒细胞凋亡相关基因bax的表达,对bcl-2的表达则无明显影响。4) 硒具有一定的抑制镉致颗粒细胞凋亡的作用,但不能完全阻断。更多还原

【Abstract】 Cadmium is a contamination of environment and industry. It has obvious toxicity for the reproductive system of female mammalian, which could cause the ovary tissue pathological change, disturbed ovulation and fecundation, lead to sterility provisional, both of the accepters of estrogen and progesterone were affected by cadmium, and the expression of genes as well.Research Aim: The effect of cadmium on the ovarian granulosa cells was defecated and the mechanism was clarified. The theory of cadmium-induced the toxicity on gonad was renewed. The theoretic evidence was provided for later study.Research Methods: Prepubertal female mice as object injected with several dose cadmium chloride after subcutaneous injected with PMSG withdrawal 48 hours. The follicular atresia was detected by pathological method, the apoptosis of granulosa cells were detected by TUNEL and agarose gel electrophoresis; the expression of bax and bcl-2 gene were detected by immunohistochemical, the antagonistic effect of selenium to cadmium was discussed by selenite natrium.Research Results: There is DNA ladder in 96-hours’ high-dose group (4mg/kg-wb) by agarose gel electrophoresis, there are no DNA ladder in 48-hours’ groups. The results showed that the apoptosis percent of granulosa cells in high, midst , low-dose groups and Se-group are 13.89%,10.76%,10.02%,5.7232% respectively after 48 hours, which are higher than control group significantly(0.6378%, P<0.01).Apoptosis percent of GCs rise obviously in 96-hours’ control group( 11.0963%).the high, midst , low-dose groups and Se-group are 20.3204%,12.2031%,12.2171%,14.9203% respectively after 96 hours. There is a significant difference between high-dose group and others, but midst and low-dose groups are similar with control group. The result showed that the apoptosis in diverse degree were occurred in every groups, the apoptosis index increase with dosage rising, there is extreme significant difference (P<0.01). The percent of atresic follicular increase with dosage rising (P<0.05).The result of expression of Bax and Bcl-2 showed that the Bax expressed widely in every groups, and it enhanced with dosage, there is extreme significant difference between experiment group and control group (P<0.01),there is not difference between withdrawal 48 hours and 96 hours. The expression of Bcl-2 was weak or negative; there is no difference among various groups.Research Conclusion: l)Cadmium can accelerate the apoptosis of GCs and is related with dose; 2)Cadmium accelerate atresic of follicular; and increase the number of interstitial gland;3)Cadmium enhance the expression of bax, and no affect obviously on bcl-2;4) Selenium restrained the effect of cadmium-induced apoptosis on granulosa cells, but it can’t be interdicted completely.更多还原