甲氧滴滴涕的子宫毒性和对子宫内膜血管生成素-1表达的影响

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论文作者签名： 导师签名： 日期：甲氧滴滴涕的子宫毒性及对子宫内膜血管生成素-1表达的影响研 究 生：杜鹃学科专业：妇产科学所在单位：第四军医大学西京医院妇产科导师：陈必良教授（主任医师）辅导教师：张建芳讲师关键词：环境内分泌干扰物；甲氧滴滴涕；子宫内膜；血管生成素-1；免疫组织化学；逆转录聚合酶链式反应中国人民解放军第四军医大学2009年05月目录缩略语表 (1)中文摘要 (3)英文摘要 (6)前言 (10)文献回顾 (12)正文 (33)实验一甲氧滴滴涕染毒雌性小鼠模型的构建及其对子宫的毒性作用331 材料 (33)2 方法 (34)3 结果 (36)4 讨论 (42)实验二甲氧滴滴涕对小鼠子宫内膜血管生成素-1蛋白表达水平的影响 (45)1 材料 (45)2 方法 (46)3 结果 (49)4 讨论 (51)实验三甲氧滴滴涕染毒对小鼠子宫内膜血管生成素-1mRNA表达水平的影响 (53)1 材料 (53)2 方法 (54)3结果 (56)4 讨论 (57)小结 (59)参考文献 (60)附图 (72)个人简历和研究成果 (77)致谢 (78)缩略语表缩略词英文全称中文全称EDC S Endocrine Disruptor Chemicals环境内分泌干扰物EMS Endometriosis子宫内膜异位症HE Hematoxylinandeosin 苏木素-伊红染色E2 Estradiol 雌二醇MXC Methoxychlor 甲氧滴滴涕OC Organochlorine 有机氯OW/BW Organ weight/Body weight 脏体比值/脏器指数PAH Polycylicaromatichydrocarbons 多环芳香烃PBS Phosphatebufferedsaline 磷酸盐缓冲液PCBs Polychlorinatedbiphenyls 聚氯联苯POP S persistent organic pollutants 持久性有机污染物SP Strepavidin peroxidase 链亲和素-过氧化物酶MDA Malondialdehyde 丙二醛CAT Catalase 过氧化氢酶DAB Diaminobenzidine 二氨基联苯POF Prematureovarianfailure 卵巢早衰HPTE 2,2-bis-(p-hydroxypheny)-1,1,1-trichloroethane2,2-双(p-羟基苯)-1,1,1-三氯乙烷1AMH Anti-Mullerianhormone 抗苗勒管激素GE Genistin 染料木黄酮ANGPT Angiopoietin 血管生成素Tie-2 Tyrosine kinase with immunoglobulinand epidermal growth factor homologydomains-2酪氨酸激酶-2VEGF Vascularendothelial growth factor 血管内皮生长因子ECS Endothelialcells 血管内皮细胞VSMCs Endometrial smooth muscle cell 子宫内膜平滑肌细胞HESCs Endometrial mesenchymal cell 子宫内膜间质细胞ACH Endometrialatypicalhyperplasia 子宫内膜复杂不典型增生EC Endometrialcancer 子宫内膜癌MPA MedroxyprogesteroneAcetate 醋酸甲羟孕酮UPI Uterineproliferationindex 子宫增殖指数 RT Reversetranscription 逆转录PCR Polymerasechainreaction 聚合酶链反应P Progesterone 孕激素AMH Anti-Mullerianhormone 抗苗勒管激素2甲氧滴滴涕的子宫毒性及对子宫内膜血管生成素-1表达的影响硕士研究生：杜鹃导师：陈必良教授辅导教师：张建芳讲师第四军医大学西京医院妇产科，西安 710032中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。

MXC及其代谢产物已经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，可以使子宫内膜增厚，质量增加，可能影响卵泡的周期性变化。

而目前有关MXC对子宫内膜血管生成的影响报道较少。

因此研究甲氧滴滴涕对子宫组织，特别是对内膜血管生成的毒性作用具有重要意义。

本研究通过构建雌性小鼠甲氧滴滴涕染毒模型，观察其对雌鼠子宫内膜血管生成素-1表达3的影响，进一步探讨MXC在子宫内膜疾病发生发展中的作用，试图为临床诊断及治疗提供新的理论依据。

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子宫的毒性作用将40只成年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MXC 50，100，200mg/(kg·d)组，连续21d天经腹腔注射MXC 染毒，染毒结束后立即处死小鼠，观察指标有：体重、子宫增殖指数；苏木素-伊红染色（Hematoxylin-Eosin Stain，HE）光镜下测量子宫内膜上皮细胞层增生高度和基质细胞层厚度；放射免疫法测定血清中E2和P的水平。

结果：①各剂量组小鼠体重成增长趋势，但无显著性差异，中高剂量组子宫内膜上皮细胞增生高度和基质细胞层厚度与对照组相比有显著性差异。

②中高剂量组子宫增殖指数随剂量增加而升高；③中、高剂量组血清E2水平①可以增加子宫重量，显著降低，P与对照组比较无显著性差异。

结论：MXC增加子宫内膜上皮细胞增生高度及基质细胞层厚度；②中高剂量的MXC可能通过影响下丘脑-垂体-卵巢轴从而使E2水平降低；③低剂量组对子宫组织未产生明显的影响。

第二部分：甲氧滴滴涕染毒对小鼠子宫内膜血管生成素-1蛋白表达水平的影响以实验一中石蜡包埋子宫组织为标本，观察指标有：免疫组化SP法和免疫荧光，显微镜下观察，计算各剂量组平均光密度；免疫组化SP法观察各剂量组子宫内膜组织ANGPT-1蛋白表达水平。

结果：①SP法显示随染毒剂量增加，中高剂量组ANGPT-1蛋白在子宫内膜中的表达明显高于对照组，平均光密度较低剂量组及对照组增加，低剂量组与对照组无显著性差异；②与分泌期相比，中高剂量组子宫内膜ANGPT-1在增殖期的表达增加更明显。

结论：①中高剂量MXC可提高小鼠子宫内膜组织血管生成素ANGPT-1蛋白的表达；②中高剂量组子宫内膜ANGPT-1在增殖期的增加4可能是呈雌激素依赖性，可能促进子宫内膜修复，也可能促进异常子宫内膜血管生成，如子宫内膜增生过长及子宫内膜癌等；在分泌期的增加可能是通过使VSMC增殖而促进螺旋动脉成熟和稳定。

第三部分：甲氧滴滴涕染毒对小鼠子宫内膜血管生成素-1mRNA表达水平的影响动物染毒途径及方法同实验一，处死各组小鼠后迅速取出子宫内膜组织，提取RNA。

采用反转录多聚酶链式反应（RT-PCR）的方法检测ANGPT-1 mRNA的表达水平，计算ANGPT-1mRNA相对含量。

结果：MXC中高剂量组ANGPT-1mRNA相对含量明显高于对照组，低剂量组与对照组表达无差异性。

结论：中高剂量的MXC可以提高小鼠子宫内膜ANGPT-1基因的转录水平，进而影响子宫内膜的生理病理过程。

关键词：环境内分泌干扰物；甲氧滴滴涕；子宫内膜；血管生成素-1；免疫组织化学；逆转录聚合酶链式反应5Effects of methoxychlor on uterine toxicity and ANGPT-1 expression of endometriumCandidate for master:Du JuanSupervisor:Prof. Chen BiliangVice-supervisor:Zhang JianfangDepartment of Obstetrics and Gynecology, Xijing HospitalFourth Military Medical University, Xi’an 710032, ChinaAbstractThere are many exogenous chemicals which can interfere the synthesis, transportation, binding, function or elimination of hormones in vivo, which maitain balance, reproduction, development and behavior in human beings. These chemicals can mimic, strengthen or inhibiti the synthesis of hormones in vivo. Under some circumstances, they may result in abnormality of tissues and organs as well as tumor. These kinds of chemicals are defined as endocrine disrupting chemicals. More and more evidence indicated that the exposure of these chemicals through occupation and enviorment would influence and change the generation function of human beings.Methoxychlor(MXC) is an organochlorine pesticide, which is widely used to be an alternative to dichloro-diphenyl-trichloroethane (DDT) for the past few years for its less toxic and rapidly metabolized and excreted in mammals. It had been confirmed methoxychlor and her metabolism could disorder the hypothalamic-pituitary-ovarian axis, and then reduced synthesis and secretion of6steroid hormone. They could increase the endometria thickness and qualities and might affect the cyclic variation of ovarian follicle. But the effects of methoxychlor on ANGPT-1 expression of endometrium in female mice had few reports at the present. For this reason, it is necessary to investigate the toxic effects of MXC on mice uterus, especially on ANGPT-1 expression of endometrium. The study was designed to analyze the effects of MXC on ANGPT-1 expression of endometrium through construction of methoxychlor poisoned mice model, which futher tried to expore the action of MXC in development and growth of pathological changes on uterus, tried to provide some useful theories for clinic diagnosis and treatment.Part 1. Construction of methoxychlor poisoned mice model and effects of methoxychlor on mice uterusMXC were administrated to forty female at doses of 0 (control), 50, 100, 200mg/(kg·d) via intraperitoneal injection for 21d, then mice were euthanized immediately. Body weights, proliferation index of uterus were observed；after Hematoxylin and eosin staining, measuring the hyperplastic hight of endometrium epithelial cell and thickness of endometrium stroma cell layer turn to light microscope; estradiol(E2) and progestereone(P) were detected with the radio immunoassays. Results: ①Increasing of body weights was not different between 4 groups after treatment；the hyperplastic hight of endometrium epithelial cell and thickness of endometrium stroma cell layer in MXC 100 and 200mg/(kg·d) groups were obviously different from the control group.②Proliferation index of uterus were gradually increasing along with MXC 100 and 200mg/(kg·d) groups which were growing. ③Estradiol (E2) were significantly decreased in 100, 200 MXC groups. There were no significant differences on serum progestereone (P) in 4 groups. Conclusion: ①MXC could7increased uterus weights and hyperplastic hight of endometrium epithelial cell and thickness of endometrium stroma cell layer. ②MXC in experimental doses might through disorder the hypothalamic-pituitary-ovarian axis and induced the decrease of E2. ③In low-dose groups there were no significant influence on uterus tissue.Part 2. Effects of methoxychlor on endometrium of ANGPT-1 proteinum expression in female miceWe used the uterus tissue embeded with paraffin just from the first experiment. Then the ANGPT-1cell were observed by electron microscope; Calculating average optical in 4 groups; The ANGPT-1 proteinum expression in uterus were examined with the SP immunohisochemical method. Results: ①The SP immunohisochemical method discovered the average optical in 100, 200mg/ (kg·d)groups was obviously higherthan that in 50mg/(kg·d)and control groups; The ANGPT-1 proteinum expression in 100, 200mg/(kg·d) groups were significantly increased on endometrium, while these dose does not change ANGPT-1 expression in 50mg/(kg·d) groups.②Compared to secretory phase, the ANGPT-1 proteinum expression in 100, 200mg/(kg·d) groups were much more increased than proliferative phase. Conclusion:①MXC in 100, 200mg/(kg·d) could increase the ANGPT-1 proteinum expression on endometrium; ②The increase on proliferative phase of ANGPT-1 proteinum expression in 100, 200mg/(kg·d)groups might display an estrogenic dependence, which might facilitate the recovery of endometrium, or might facilitate the abnomal angiogenesis of endometrium, e.g excess hyperplasia endometrium and endometrial cancer; but the increase on secretory phase might through VSMC proliferation to facilitate maturation stabilization of helicine artery.Part 3. Effects of methoxychlor on endometrium of ANGPT-1 mRNA expression in female mice8We erected the model on intraperitoneal injection MXC just like the first experiment. Then mice were euthanized, then collecting their endometrium and extracting RNA，Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of ANGPT-1 mRNA in mice uterus, calculating relative amount of ANGPT-1 mRNA. Results:MXC in 100, 200mg/(kg·d) groups could obviously super-normal endometrium, but there were no obviously differences in MXC in 50mg/(kg·d)groups. Conclusion: MXC in 100, 200mg/(kg·d)groups could increase ANGPT-1 mRNA expression in female mice on endometrium, and then affected the process of physiology and pathology.Key words: Environmental endocrine disruptors; Methoxychlor;Endometrium;Angiopoietin-1;Immunohistochemistry; Reverse transcriptionpolymerase chain reaction9前言日益严重的环境污染已经对人类健康生存和持续繁衍构成严重的威胁。