【作者】 付远辉；

【导师】 吴逸明； 陈小玉；

【作者基本信息】 郑州大学 ， 劳动卫生与环境卫生学， 2004， 硕士

【摘要】 近年来越来越多的研究表明，人类和野生动物的不良健康效应可能与环境中的某些化学物质有关。由于这些化学物质能干扰内分泌功能，故称为环境内分泌干扰物（environmental endocrine disruptors，EEDs），其中环境雌激素（Environmental estrogens，EEs）是较为重要的一类环境内分泌干扰物，他能够引起肿瘤发病率增高、生殖功能损害、神经和免疫功能的损伤。关于环境雌激素样物质的研究，目前多集中在单一物质较大剂量对机体的影响，而环境中的化合物往往难以达到这个剂量；且绝大多数环境雌激素样物质的雌激素活性十分微弱，仅为E₂的1／1000至1／10000，如单独研究一种雌激素活性较弱的物质的雌激素样作用（特别是不具蓄积作用的环境雌激素样物质），往往会发现他们对生物体几乎不产生任何损害作用。事实上，环境雌激素样物质常以混合物的形式存在于环境中，它们往往是以低剂量混合进入机体，对机体产生联合作用，因而其危害往往被低估了。随着对环境雌激素样物质的深入研究，环境雌激素样物质的联合作用已引起关注，目前关于环境雌激素样物质联合作用的研究，郑州人学医学院2004届硕!论文水与锡雌激素样联合作用的实验研究主要集中在杀虫剂类（如:狄氏剂、硫丹、毒杀芬等）和某些工业化学物（如:PCBs、双酚A等）等，重金属类雌激素样物质的联合作用尚未见报道。HgC12和cdCI:是重要的工业化合物，他们进入人体后，一部分经代谢排出，但大部分能够在体内蓄积，近年来己有报道认为汞和福是环境雌激素样物质。为此，本研究用去卵巢大鼠子宫增重试验、大鼠子宫内膜增殖实验、MCF一7人乳腺癌细胞增殖试验等，对低剂量HgC12与 CdCI:雌激素样联合作用进行初步探讨:并用放射性配基结合分析法测定去卵巢大鼠子宫雌激素受体数量，进一步探讨HgCI:与cdcl:是通过何种途径显示雌激素样作用。方法 1.去卵巢大鼠子宫增重试验参照Diel介绍的方法，给大鼠行双侧去卵巢手术，于术后第sd随机分11组，每组6只，分别为阴性对照组（蒸馏水），HgCI:低、中、高剂量组（o.04mg/kg、0.20mg/kg和1 .oomg/kg），CdCI:低、中、高剂量组（0.08mg/kg、0.40mg/kg和2.00mg/kg），（HgCIZ+CdC12）低、中、高剂量组[（0.04+0.08）mg/kg、（ 0.04+0.40）mg/kg和（0.04+2.00）mg/kg]，阳性对照组（E z0.08m留kg，以花生油为溶剂），给药容积为2.oml/kg体重，每天腹腔注射一次，连续3d。最后一次给药后24h，称重，颈椎脱臼处死大鼠，处死前2h每只大鼠腹腔注射秋水仙素4mg/kg，使细胞分裂停留于有丝分裂中期。迅速取出子宫，剔除周围结缔组织和脂肪，吸干子宫表面血渍和子宫内液，于万分之一天平上称量。 2.去卵巢大鼠子宫内膜增殖实验于子宫中段垂直于子宫角长轴朝子宫体方向切取约l。m子宫组织，经Bouin‘s液固定，在24h内，用体积分数为70%乙醇洗涤直至苦味酸的黄色消失。常规石蜡包埋，一半切片进行常规HE染色;另一半进行硝酸银染色;石蜡切片经二甲苯脱色后，逐级乙醇脱水，自来水和去离子水冲洗，滴加新鲜配制的Ag一NORs银染工作液于切片上，室温（25℃）避光0.5h，去离子水冲洗，逐级乙醇脱水后用二甲苯透明，中性树脂封片。郑州大学医学院2004届硕十论文汞与锡雌激素样联合作用的实验研究 3.MCF一7人乳腺癌细胞增殖实验按照Perez等的方法并做一些改良:细胞采用开放式单层贴壁培养，培养液为RPMI一1 640（含体积分数为10ryo的小牛血清，30U/IOOml胰岛素），培养条件为37℃，5%C仇，100%相对饱和湿度。加受试物前先将细胞用PBS洗涤，然后换为无酚红RPMI一1640培养液（含8W0经活性碳一葡聚糖处理的胎牛血清），继续培养2d，以耗尽细胞内储存的雌激素。生长至80%融合时，用0.25%胰蛋白酶消化，以10000个细胞/ml接种于96孔板，每孔接种200 pl，各剂量均设4个平行孔。贴壁培养24h后，弃孔内培养液，加入200时含有不同剂量受试物、溶剂对照以及阳性物的不含酚红的RPMI一1 640培养基。每3d换液1次，培养6d。在第6d细胞增殖的指数期检测细胞增长情况。本实验采用MTT比色法检测总蛋白含量。培养6d后弃上清，每孔加入噬吟兰（MTT）25曰，放置在培养箱中孵育4h，小心吸取干净孔内液体后，每孔加入二甲基亚矾（DMSO）1 50 pl，振荡10min后，用酶联免疫检测仪于490nm波长处测定吸光值。4.去卵巢大鼠子宫雌激素受体测定实验用预冷生理盐水冲洗大鼠子宫增重试验所得子宫，称重，计算所需组织抽提液的体积，剪碎，按1:10（mg/ml）加入预冷的抽提缓冲液，在冰浴条件下用玻璃匀浆器匀浆，15000rpm，4℃离心30min，上清液即为胞浆液。用考马斯亮蓝G一250/BSA法测定胞浆液中蛋白含量，放射性配基结合分析法测定大鼠子宫雌激素受体含量。采用10nmol/L3H-雌二醇作单点饱和分析，葡聚糖一活性炭（Dcc）法分离游离的和结合的3H一EZ后，将上清液倒入sml的闪烁液中，用F小2，01G双道液体闪烁计数器计数。结果 1.去卵巢大鼠子宫增重试验中，HgCI:和CdCI:各剂量组均能诱导去卵巢大鼠子宫增殖，子宫脏器系数随着染毒剂量的增加而递增，且呈剂量一效应关系，但与阴性对照组相比，仅高更多还原

【Abstract】 Currently, more and more studies have reported that a few adverse health effects on human have been attributed to some chemicals existing in environment. Because these chemicals can affect the function of endocrine, they are called environmental endocrine disruptors(EEDs). Being one of the most important environmental endocrine disrupters , Environmental estrogens (EEs) have a few adverse health effects on human such as tumors dependent of hormone, abnormalities of immunity function, nerve system and reproductive and developmental function .Most of the studies about environmental estrogen focused effect of the single chemical with high dose on human,but it is difficult to up to this dose for the single chemical.The estrogenic activity of mostof EEs is only 1/ 1000 to 1/ 10000 of the activity of E2.If study the estrogenic effect of the chemical with weakly estrogenic activity ,you can find it hardly has harm to organism.Now many scientist had focused their studies on the joint effect of environmental estrogens. Most of studies on joint effect of environmental estrogens focused on insecticide and techchemical ,but there is not study on estrogenic joint effect of metal.some studies reported that Cadmium Chloride and Mercury Chloride have the estrogen-like effect ,so this study will evaluate estrogenic joint effect of Cadmium Chloride and Mercury Chloride with proliferation assay on MCF-7 human breast cancer cells, uterotrophic assay in ovariectomized SD rats and proliferation assay of mucous membrane. Methods1. Uterotrophic assay : Female SD rats were ovariectomized,8 days later, the rats were divided into 11 groups at random, which is negative control group(distilled water), low Mercury Chloride group (0.04mg/kg) .middle dose group (0.20mg/kg), high dose group(1.00mg/kg), low dose Cadmium Chloridegroup(0.08mg/kg),middle dose group(0.40mg/kg) , high dose group(2.00mg/kg), low dose (Mercury Chloride + Cadmium Chloride) group[(0.04+0.08)mg/kg] , middle dose group[(0.04+0.40)mg/kg], high dose group[(0.04+2.00)mg/kg] and positive control group(80g/kg 17 3 -estradiol, solvent oil). The dose of 2.0ml/kg body weight was given to animals, and administration was given per day by intraperitonal injection for 3 days. All animals were injected Colchicine with the dose of 4mg/Kg. All animals’ body weights were measured 24 hours after final injection, then all animals were sacrificed. Uterus were removed quickly, fat and connective tissue were peeled off , blood and liquid of uterus were absorbed with tissue, then uterus weights were measured.2. Proliferation assay of cell of mucous membrane: Cut off a part of uterus plumbing horn of uterus at the middle part of uterus,and fox the uterus in the Bouin’s ,then wash the uterus with 70% alcohol till destaining of yellow of the bitter acid. The uterus tissues was embeded with paraffin regularly, half of tissues are stained by HE,the half tissues are stained by silver nitrate .Put the slices in Dimethylbenzene,alcohol,tap water,distilled water in turn, then add the fresh prepared Ag-NoRs on the slices. After a half hour, put the slices in distilled water,alcohol,dimethybenzene in turn,then seal with neutral gum.3. Proliferation assay of MCF-7 human breast cancer cells: each well seeded cells(1 104 cells/ml) in 96 wells plates in media of RPMI-1640 supplemented with 8% dextran/Charcoal-treated FBS and insulin(30u/mL). Cells were incubated at the environment of 37 C,5%CO2 for 24 hours after they being adhered to walls, thenremoved the medium, and added 200 u 1 of RPMI-1640 medium with different doses of chemicals described above , blank media as negative control and 17 -estradiol as positive control. Each dose was repeated 4 times arranged at parallel wells. Media were changed once 3 days interval and were removed after 6 days , 25 1 of thiazolyl blue(MTT) was added to each well, then plates were put into incubator for 4 hours, then MTT was removed and 150 1 DMSO was added to each well.更多还原