甲氧滴滴涕染毒对雌性SD大鼠的性腺毒性及其机制研究

发布时间：2019-05-18 09:26

【摘要】： 外环境中存在许多能模拟或干扰机体内分泌功能的化学物,影响体内激素的合成、分泌、运输、结合、作用、代谢或消除等环节,从而对个体的生殖、发育以及生物行为产生影响,表现出拟天然激素或抗天然激素的作用,这一类化合物统称为环境内分泌干扰物(Endocrine Disruptor Chemicals,EDCs),可以对人体的生殖健康产生严重的影响。生殖健康包括了男女两个方面,由于女性的生殖过程远较男性复杂,但在有关生殖健康的研究中,有关环境污染物对女性健康影响的资料与男性相比相对较少。而女性在人类生殖的过程中,不仅提供繁衍生命的生殖细胞,还提供孕育生命的母体环境,因此保护女性的生殖健康对人类的繁衍具有重要意义。甲氧滴滴涕(methoxychlor,MXC)是一种替代滴滴涕的有机氯杀虫剂,其杀虫效果明显,生物毒性相对较低,在许多国家得到广泛应用。然而,近几年的研究表明,甲氧滴滴涕是确定的环境内分泌干扰物,可对雌性和雄性生殖内分泌产生影响。很多研究表明MXC对雄性生殖系统有破坏作用,但是对于雌性研究的较少。本课题通过构建雌性SD大鼠染毒模型,研究MXC对雌性大鼠性腺的毒性作用及卵巢抗氧化功能的影响,并观察了MXC对卵巢和子宫ER蛋白表达及垂体FSH表达的调节机制,为深入阐明MXC对雌性动物生殖功能的影响提供理论和实验依据。第一部分:甲氧滴滴涕染毒雌性SD大鼠模型的构建及对生殖内分泌的干扰作用使用雌性SD大鼠作为实验动物,3月龄,随机分为4组,每组10只,染毒剂量分别为0(对照)、16(低剂量)、32(中剂量)、64(高剂量)mg/(kg?d),对照组给予等剂量芝麻油,所有实验大鼠均通过腹腔注射染毒,连续20d。每天用阴道脱落细胞涂片法连续观察并记录动情周期。染毒结束后于动情期将大鼠处死,观察指标包括体重,卵巢、子宫的脏器系数;放射免疫法测定血清中E2、P、FSH和LH的水平。免疫组化SP法观察雌激素受体(ER)在子宫和卵巢组织中的表达,免疫荧光染色观察FSH在垂体的表达;运用光镜和电镜观察卵巢组织和细胞超微结构的变化,计数各级卵泡及黄体数目。结果表明:①染毒前后各组体质量增加未见显著差异;②中高剂量染毒组动情期延长,周期数减少;③中高剂量组卵巢体质量及卵巢指数显著降低;④中、高剂量染毒组血清FSH和E2水平显著降低,P和LH与对照组比较无显著性差异;⑤中高剂量组闭锁卵泡显著增加,且次级卵泡闭锁率显著升高;⑥各组子宫内膜和卵巢ER蛋白的表达无显著性差异;⑦中高剂量组垂体FSH表达水平显著降低。结论:①甲氧滴滴涕可以使动情周期延长,卵泡闭锁增加,对雌性性腺产生毒性;②实验剂量的甲氧滴滴涕可以引起下丘脑-垂体-卵巢轴紊乱,降低垂体FSH表达水平,但并不是通过改变卵巢和子宫ER蛋白表达水平引起雌性性腺毒性的;③低剂量组未对雌性性腺产生明显的影响。第二部分:甲氧滴滴涕染毒对SD大鼠卵巢抗氧化系统功能的影响实验动物染毒模型构建同实验一,将雌性SD大鼠随机分为MXC 0、32、64mg/(kg?d)及MXC 64 mg+维生素E 20 mg/(kg?d)组。20 d后处死大鼠,用分光光度法测定其卵巢中超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)的活力以及丙二醛(MDA)的含量;用RT-PCR法观察卵巢组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)以及过氧化氢酶(CAT)等抗氧化酶mRNA的表达。结果表明:①中高剂量MXC组SOD、GSH活性显著下降,MDA含量升高,MXC64mg+维生素E20mg组SOD、GSH活性及MDA含量与对照组比较无显著性差异;②RT-PCR结果显示MXC中高剂量组卵巢中抗氧化酶(SOD、GSH-Px、CAT)mRNA表达均显著降低,MXC64mg+维生素E20mg组抗氧化酶mRNA表达与对照组比较无显著性差异。结论:①MXC可以对卵巢抗氧化系统产生影响,造成抗氧化酶活性降低,脂质过氧化增强;②维生素E可以对MXC引起的氧化应激损伤产生拮抗作用;③中高剂量组MXC引起卵泡闭锁可能是通过氧化应激产生。
[Abstract]:The external environment has many chemical substances which can simulate or interfere with the endocrine function of the body, and influence the synthesis, secretion, transportation, binding, action, metabolism or elimination of the hormone in the body, thus affecting the reproduction, development and biological behavior of the individual, The effect of the quasi-natural hormone or the anti-natural hormone is shown, and the compounds are collectively referred to as environmental endocrine disruptors (EDCs), which can have a serious effect on the reproductive health of the human body. Reproductive health includes both men and women, as women's reproductive processes are far more complex than men, but in research on reproductive health, information on the impact of environmental pollutants on women's health is relatively small compared to men. In the process of human reproduction, women not only provide the germ cells that reproduce the life, but also provide the maternal environment for the life, so the protection of the reproductive health of women is of great significance to the reproduction of human beings. methoxychlor (MXC) is a kind of organochlorine pesticide, which is an alternative to DDT, its insecticidal effect is obvious, the biological toxicity is relatively low, in many countries In recent years, however, studies have shown that methoxychlor is a defined environmental endocrine disruptor that can be used in female and male reproduction The effect of MXC on the male reproductive system has been shown in many studies. In this study, the effects of MXC on the sexual function of the gonad in female rats and the function of the anti-oxidation of the ovary were studied, and the expression of the MXC on the ovary and the uterus of the uterus was studied. And the regulation mechanism of the expression of the pituitary FSH is a mechanism for further elucidating the reproductive work of the MXC to the female animals. Theoretical and experimental basis for the effects of the energy provided. Part one: methoxychlor-exposed female The construction of SD rat model and the effect on reproductive endocrinology use female SD rats as experimental animals and 3 months of age, and were randomly divided into 4 groups. 10 of each group,0 (control),16 (low dose),32 (middle dose),64 (high dose) mg/ (kg? d), control group, etc. Doses of sesame oil, all of the experimental rats were exposed by intraperitoneal injection and were continuous 20 d. The cell smear method was used to observe and record the estrus cycle on a daily basis. The rats were sacrificed after the end of the exposure. The observation index included the body weight, the ovary and the uterus. The levels of E2, P, FSH, and LH in serum were determined by radioimmunoassay. The expression of estrogen receptor (ER) in the uterus and ovary was observed by immunohistochemical SP method, and the expression of FSH in the pituitary was observed by immunofluorescent staining. The changes of the ultrastructure of the ovarian tissues and the cells were observed, and the numbers of the follicles and the corpus luteum at all levels were counted. The results showed that there was no significant difference in the quality of the body before and after the oral administration. The levels of serum FSH and E2 in the high-dose group were significantly decreased in the middle and high dose groups, and there was no significant difference between the levels of P and LH and the control group. There was no significant difference in the level of the expression of FSH in the high-dose group. Conclusion: DDT can prolong the estrus cycle, increase the atresia of the follicle, and produce the toxicity to the gonad of the female. in ord to cause that hypothalamic-pituitary-ovarian axis disorder to reduce the level of pituitary FSH expression, but not by changing the expression level of the ovary and the uterus ER protein, The female gonad was toxic; the low-dose group did not have a significant effect on the female gonads. The second part: The effect of methoxychlor on the function of the anti-oxidation system in SD rats was the same as that of the experiment. The female SD rats were randomly divided into MXC 0,32,64 mg. The activity of superoxide dismutase (SOD) and reduced glutathione (GSH) in the ovary was determined by spectrophotometry. and the content of the malondialdehyde (MDA) is determined; the superoxide dismutase (SOD) in the ovarian tissue is observed by the RT-PCR method, The results showed that the activity of SOD and GSH in the high-dose MXC group decreased significantly, and the content of MDA increased, and the content of SOD, GSH and MDA in the group of MXC64mg + vitamin E20mg was not significantly higher than that of the control group. The results of RT-PCR showed that the expression of antioxidant enzymes (SOD, GSH-Px, CAT) in the ovary of the high-dose group of MXC decreased significantly. Conclusion: The expression of anti-oxidation enzyme in the group of MXC64mg + vitamin E20mg is not significantly different from that of the control group. Conclusion: The effect of XMXC on the anti-oxidation system of the ovary, the decrease of the activity of anti-oxidation enzyme and the enhancement of lipid peroxidation, and the effect of vitamin E on the MXC
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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