线粒体损伤和细胞凋亡在甲氧滴滴涕卵巢毒性中的作用

发布时间：2018-05-10 12:06

本文选题：环境内分泌干扰物 + 甲氧滴滴涕　；参考：《第四军医大学》2008年硕士论文

【摘要】： 环境内分泌干扰物(Endocrine Disrupting Chemicals,EDCs)被定义为对维持自身平衡、生殖、发育和行为的体内激素的合成、传输、键合、作用或清除有干扰作用的外源性物质,这些物质能够模拟、强化或抑制激素合成的作用,在某些情况下,可能引发组织或器官功能异常和肿瘤发生。由于职业和环境因素使人类越来越多地暴露于这类干扰物中,严重影响人类的生殖功能和身体健康。作为环境内分泌干扰物之一的甲氧滴滴涕(methoxychlor,MXC),是近年来被广泛应用的、代替滴滴涕的有机氯杀虫剂,与滴滴涕相比具有在哺乳动物体内代谢快,易排泄、毒性低等特点。然而,甲氧滴滴涕被证实可引起雌性大鼠下丘脑-垂体-卵巢性腺轴紊乱,合成、分泌甾体激素减少、动情期延长、雄性生殖系统异常,等一系列毒性损害,因此研究甲氧滴滴涕对卵巢组织的毒性作用及其作用机制具有重要意义。本研究通过构建雌性小鼠甲氧滴滴涕染毒模型,观察其对雌鼠卵巢组织的毒性损害作用,探讨参与这一过程的细胞凋亡途径及线粒体损伤机制。本实验分三部分: 第一部分:甲氧滴滴涕染毒雌性小鼠模型的构建及其对卵巢的毒性作用将40只32日龄雌性昆明小鼠适应性饲养7日后,随机分为溶剂芝麻油组(对照组)及MXC16,32,64mg/(kg·d)组,连续20天经腹腔注射染毒MXC,染毒结束后于动情期处死小鼠,观察指标有:体重、卵巢脏器指数;苏木素-伊红染色(Hematoxylin and eosin,HE)光镜下计数各剂量组卵泡闭锁数目,计算卵泡闭锁百分率;放射免疫法测定血清中E2、P、FSH和LH的水平。结果:①各剂量组小鼠体重无显著性差异,中高剂量组卵巢质量逐渐下降,脏器指数显著降低;②中高剂量组闭锁卵泡百分率随剂量增加而升高;③中、高剂量组血清FSH和E2水平显著降低,P和LH与对照组比较无显著性差异。结论:①MXC可以降低卵巢质量,增加卵泡闭锁;②中高剂量的MXC可以引起实验剂量的甲氧滴滴涕可以引起下丘脑-垂体-卵巢轴紊乱,导致FSH和E2水平降低;③低剂量组对卵巢组织未产生明显的影响。第二部分:甲氧滴滴涕染毒对颗粒细胞凋亡机制的研究以实验一中石蜡包埋卵巢组织为标本,观察指标有:脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(TUNEL法)染色,荧光显微镜下观察、计数各剂量组卵巢组织中颗粒细胞凋亡的情况;透射电镜观察卵巢颗粒细胞超微结构改变;免疫组化SP法观察各剂量组卵巢组织Bax、Bcl-2蛋白表达水平。结果:①TUNEL法显示随染毒剂量增加,中高剂量组凋亡细胞数较低剂量组及对照组增加;②透射电镜见中高剂量组颗粒细胞内出现凋亡小体、胞浆空泡等典型凋亡形态学改变③中高剂量组Bax蛋白在卵巢中的表达明显高于对照组,Bcl-2蛋白的表达各组间无显著性差异。结论:①中高剂量的MXC可以引起明显的颗粒细胞凋亡;②中高剂量的MXC可提高小鼠卵巢组织促凋亡蛋白Bax的表达,发挥其诱导颗粒细胞凋亡的作用。第三部分:甲氧滴滴涕对卵巢组织线粒体损伤机制的研究动物染毒途径及方法同实验一,处死各组小鼠后迅速取出双侧卵巢制备组织匀浆,经多次低温高速离心提取出卵巢组织线粒体后,荧光分光光度计测各剂量组卵巢线粒体活性氧簇(reactive oxygen species,ROS)、膜电势的变化;紫外分光光度法检测线粒体呼吸链复合物Ⅰ活性变化。结果:卵巢组织线粒体膜电势,线粒体呼吸链复合物Ⅰ活性在中高剂量组低于对照组,线粒体ROS水平则高于对照组。结论:中高剂量的MXC可以通过降低线粒体膜电势、抑制线粒体复合物I活性及增加活性氧的产生诱导线粒体发生氧化应激,从而促进颗粒细胞凋亡、卵泡闭锁。
[Abstract]:Environmental endocrine disruptors (Endocrine Disrupting Chemicals, EDCs) are defined as the synthesis, transmission, bonding, action, or removal of exogenous substances that have interfering effects on the maintenance of homeostasis, reproduction, development and behavior. These substances can mimic, strengthen or inhibit the role of hormone synthesis, in some cases, may be triggered. Tissue or organ dysfunction and tumorigenesis. Due to occupational and environmental factors, more and more human exposure to this kind of interferon can seriously affect human reproductive and physical health.
Methoxychlor (MXC), one of the environmental endocrine disruptors, is widely used in recent years as an organochlorine insecticide instead of DDT. Compared with DDT, it has the characteristics of fast metabolism, excretion and low toxicity in mammals. However, methoxy DDT has been proved to cause the hypothalamus hypophysis ovum in female rats. Nesting gonadal axis disorder, synthesis, secretion of steroid hormones, prolonged estrus, abnormal male reproductive system and a series of toxic damage, therefore, it is of great significance to study the toxicity and mechanism of methoxy DDT to ovarian tissue. The toxic effects of weave on apoptosis and the mechanism of mitochondrial damage involved in this process were discussed.
The experiment is divided into three parts:
Part I: the construction of a methoxy DDT infected female mouse model and its toxicity to the ovary.
After feeding 40 32 day old female Kunming mice for 7 days, they were randomly divided into the solvent sesame oil group (control group) and MXC16,32,64mg/ (kg. D) group. The mice were injected into the abdominal cavity for 20 days, and the mice were killed at the end of the estrus. The indexes were: body weight, ovarian organ index, and hematoxylin eosin (Hematoxylin and eosin, HE) light microscopy. The number of follicle atresia in each dose group was counted and the percentage of follicle atresia was calculated. The levels of E2, P, FSH and LH in serum were measured by radioimmunoassay. Results: (1) there was no significant difference in weight of the mice in each dose group, the quality of the ovary decreased gradually and the organ index decreased significantly in the middle and high dose group, and the percentage of the atresia follicle in the middle and high dose group increased with the increase of dose. The level of serum FSH and E2 in high dose group decreased significantly, and there was no significant difference between P and LH in the control group. Conclusion: (1) MXC can reduce the quality of the ovary and increase the follicle atresia, and the high dose of MXC can cause the experimental dose of DDT to cause the hypothalamus pituitary ovarian axis disorder, which leads to the decrease of FSH and E2 level; (3) low level of FSH and E2 The dose group did not have a significant effect on the ovarian tissue.
The second part: the mechanism of the apoptosis of granulosa cells exposed to methoxy DDT.
The tissue of paraffin embedded ovary in experimental one was used as a specimen, and the observation indexes were: deoxyribonucleotide terminal transferase mediated nick end labeling technique (TUNEL) staining and fluorescence microscope observation to count the apoptosis of granulosa cells in the ovarian tissue of each dose group; the ultrastructural changes of ovarian granulosa cells were observed by electron microscope; The expression of Bax and Bcl-2 protein in the ovarian tissue of each dose group was observed by the histochemical SP method. Results: (1) the number of apoptotic cells in the middle and high dose group was increased with the increase of the dose in the middle and high dose group, and the number of apoptotic bodies in the medium and high dose group, and the typical morphological changes of the cytoplasm vacuoles in the middle and high dose group were higher than that in the middle and high dose group. The expression of Bax protein in the dose group was significantly higher than that in the control group. There was no significant difference in the expression of Bcl-2 protein in the control group. Conclusion: (1) the high dose of MXC can cause obvious apoptosis of granulosa cells, and the high dose of MXC can increase the expression of the apoptosis protein Bax of the mouse ovarian tissue and play its role in inducing the apoptosis of granulosa cells.
The third part: the mechanism of methoxy DDT on mitochondria damage in ovarian tissue.
The methods and methods of animal poisoning were the same as experiment one. After the mice were killed, the ovariectomized tissue homogenates were taken out quickly and the mitochondria of the ovary were extracted by multiple cryogenic high speed centrifugation. The changes of the reactive oxygen species, ROS, the membrane potential of the ovarian mitochondria were measured by the fluorescence spectrophotometer, and the ultraviolet spectrophotometry was used. Mitochondrial respiratory chain complex I activity changes. Results: mitochondrial membrane potential of ovarian tissue, mitochondrial respiratory chain complex I activity in the middle and high dose group is lower than the control group, the mitochondrial ROS level is higher than the control group. Conclusion: high dose of MXC can reduce the mitochondrial membrane potential, inhibit the activity of mitochondrial complex I and increase the activity. The generation of sex oxygen induces oxidative stress in mitochondria, thereby promoting apoptosis of granulosa cells and follicular atresia.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R711;R363

【参考文献】