本文選題：環(huán)境內(nèi)分泌干擾物 + 甲氧滴滴涕　； 參考：《第四軍醫(yī)大學(xué)》2008年碩士論文

【摘要】： 環(huán)境內(nèi)分泌干擾物(Endocrine Disrupting Chemicals,EDCs)被定義為對維持自身平衡、生殖、發(fā)育和行為的體內(nèi)激素的合成、傳輸、鍵合、作用或清除有干擾作用的外源性物質(zhì),這些物質(zhì)能夠模擬、強化或抑制激素合成的作用,在某些情況下,可能引發(fā)組織或器官功能異常和腫瘤發(fā)生。由于職業(yè)和環(huán)境因素使人類越來越多地暴露于這類干擾物中,嚴(yán)重影響人類的生殖功能和身體健康。 作為環(huán)境內(nèi)分泌干擾物之一的甲氧滴滴涕(methoxychlor,MXC),是近年來被廣泛應(yīng)用的、代替滴滴涕的有機(jī)氯殺蟲劑,與滴滴涕相比具有在哺乳動物體內(nèi)代謝快,易排泄、毒性低等特點。然而,甲氧滴滴涕被證實可引起雌性大鼠下丘腦-垂體-卵巢性腺軸紊亂,合成、分泌甾體激素減少、動情期延長、雄性生殖系統(tǒng)異常,等一系列毒性損害,因此研究甲氧滴滴涕對卵巢組織的毒性作用及其作用機(jī)制具有重要意義。本研究通過構(gòu)建雌性小鼠甲氧滴滴涕染毒模型,觀察其對雌鼠卵巢組織的毒性損害作用,探討參與這一過程的細(xì)胞凋亡途徑及線粒體損傷機(jī)制。 本實驗分三部分: 第一部分:甲氧滴滴涕染毒雌性小鼠模型的構(gòu)建及其對卵巢的毒性作用 將40只32日齡雌性昆明小鼠適應(yīng)性飼養(yǎng)7日后,隨機(jī)分為溶劑芝麻油組(對照組)及MXC16,32,64mg/(kg·d)組,連續(xù)20天經(jīng)腹腔注射染毒MXC,染毒結(jié)束后于動情期處死小鼠,觀察指標(biāo)有:體重、卵巢臟器指數(shù);蘇木素-伊紅染色(Hematoxylin and eosin,HE)光鏡下計數(shù)各劑量組卵泡閉鎖數(shù)目,計算卵泡閉鎖百分率;放射免疫法測定血清中E2、P、FSH和LH的水平。結(jié)果:①各劑量組小鼠體重?zé)o顯著性差異,中高劑量組卵巢質(zhì)量逐漸下降,臟器指數(shù)顯著降低;②中高劑量組閉鎖卵泡百分率隨劑量增加而升高;③中、高劑量組血清FSH和E2水平顯著降低,P和LH與對照組比較無顯著性差異。結(jié)論:①MXC可以降低卵巢質(zhì)量,增加卵泡閉鎖;②中高劑量的MXC可以引起實驗劑量的甲氧滴滴涕可以引起下丘腦-垂體-卵巢軸紊亂,導(dǎo)致FSH和E2水平降低;③低劑量組對卵巢組織未產(chǎn)生明顯的影響。 第二部分:甲氧滴滴涕染毒對顆粒細(xì)胞凋亡機(jī)制的研究 以實驗一中石蠟包埋卵巢組織為標(biāo)本,觀察指標(biāo)有:脫氧核糖核苷酸末端轉(zhuǎn)移酶介導(dǎo)的缺口末端標(biāo)記技術(shù)(TUNEL法)染色,熒光顯微鏡下觀察、計數(shù)各劑量組卵巢組織中顆粒細(xì)胞凋亡的情況;透射電鏡觀察卵巢顆粒細(xì)胞超微結(jié)構(gòu)改變;免疫組化SP法觀察各劑量組卵巢組織Bax、Bcl-2蛋白表達(dá)水平。結(jié)果:①TUNEL法顯示隨染毒劑量增加,中高劑量組凋亡細(xì)胞數(shù)較低劑量組及對照組增加;②透射電鏡見中高劑量組顆粒細(xì)胞內(nèi)出現(xiàn)凋亡小體、胞漿空泡等典型凋亡形態(tài)學(xué)改變③中高劑量組Bax蛋白在卵巢中的表達(dá)明顯高于對照組,Bcl-2蛋白的表達(dá)各組間無顯著性差異。結(jié)論:①中高劑量的MXC可以引起明顯的顆粒細(xì)胞凋亡;②中高劑量的MXC可提高小鼠卵巢組織促凋亡蛋白Bax的表達(dá),發(fā)揮其誘導(dǎo)顆粒細(xì)胞凋亡的作用。 第三部分:甲氧滴滴涕對卵巢組織線粒體損傷機(jī)制的研究 動物染毒途徑及方法同實驗一,處死各組小鼠后迅速取出雙側(cè)卵巢制備組織勻漿,經(jīng)多次低溫高速離心提取出卵巢組織線粒體后,熒光分光光度計測各劑量組卵巢線粒體活性氧簇(reactive oxygen species,ROS)、膜電勢的變化;紫外分光光度法檢測線粒體呼吸鏈復(fù)合物Ⅰ活性變化。結(jié)果:卵巢組織線粒體膜電勢,線粒體呼吸鏈復(fù)合物Ⅰ活性在中高劑量組低于對照組,線粒體ROS水平則高于對照組。結(jié)論:中高劑量的MXC可以通過降低線粒體膜電勢、抑制線粒體復(fù)合物I活性及增加活性氧的產(chǎn)生誘導(dǎo)線粒體發(fā)生氧化應(yīng)激,從而促進(jìn)顆粒細(xì)胞凋亡、卵泡閉鎖。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R711;R363

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