【作者】 芦军萍；

【导师】 蔡德培； 陈秉衡； 郑力行； 季志英；

【作者基本信息】 复旦大学 ， 中西医结合临床， 2006， 博士

【摘要】 目的：探讨环境内分泌干扰物（Environmental endocrine disruptors，EEDs）与性早熟发病的关系，制定针对EEDs拟雌激素作用的以中药为主的治疗方案，并深入研究中药治疗的作用机理。方法：（1）运用反相高效液相色谱法检测73例性早熟患儿及年龄、性别匹配的38例正常儿童血清中数种有代表性的EEDs(包括：洗涤剂降解产物中的4-壬基酚（4-nonylphenol，4-NP）、有机氯农药DDT的代谢产物中的二氯二苯二氯乙烯（1，1-dichloro-2，2，bis（p-chlorophenyl）ethylene，p，p’-DDE）、塑料添加剂中的邻苯二甲酸-2-乙基己酯（di-2-ethylhexyl phthalate，DEHP）的含量；同时测定性早熟患儿的靶器官发育指标，包括子宫、卵巢体积，骨密度及血清雌二醇E₂水平。对两组儿童的EEDs血清含量进行分析，并将血清EEDs含量与靶器官发育指标进行相关性分析及多元线性回归分析。（2）以21d雌性SD大鼠为实验对象，选取4-NP、双酚A（bisphenol A，BPA）、DEHP为染毒物质，均设高中低剂量，并将4-NP和BPA以低剂量混合喂饲连续三天，于末次给药后24h处死。以子宫湿重、脏器系数、子宫内膜及平滑肌增生程度和细胞增殖核抗原（proliferating cell nuclear antigen，PCNA）的蛋白表达作为检测指标，采用单因素方差分析方法进行统计。（3）制定针对EEDs拟雌激素作用的中药治疗方案，治疗受EEDs暴露的性早熟患儿及染毒动物，以上述生殖器官及骨骼发育水平作为检测指标；并采用实时荧光定量聚合酶链式反应（real-time FQ-PCR）及免疫组织化学方法（immunohistochemistry，IHC）检测靶器官雌激素受体α（estrogen receptor alpha，ERα）、雌激素受体β（estrogen receptor beta，ERβ）、表皮细胞生长因子受体（epidermal growth factor receptor，EGFR）和胰岛素样生长因子-1受体（insulin-like factor-1 receptor，IGF-1R）的基因和蛋白的表达情况，采用单因素方差分析及t检验进行统计分析。结果：（1）每一例正常儿童血清中均检测到一定含量的p，p’-DDE（14.93～40.39ng/ml），部分正常儿童血清中检测到一定含量的4-NP及DEHP（ND～6.77ng/ml，ND～17.61ng/ml）。性早熟患儿血清中4-NP含量（ND～16.68ng/ml）、p，p’-DDE含量（40.03～91.68ng/ml）及DEHP含量（ND～64.71ng/ml）显著高于正常儿童（P＜0.01）。性早熟患儿血清中4-NP与子宫体积、卵巢体积、骨密度均呈显著正相关（r=0.394，0.286，0.237；P＜0.01）；p，p’-DDE与子宫体积呈显著正相关（r=0.306；P＜0.01）。4-NP对子宫体积的作用强度明显高于p，p’-DDE。（2）染毒动物实验结果显示，高中剂量的4-NP（200mg/kg，100mg/kg）和BPA（600mg/kg，400mg/kg）能够使染毒动物的子宫湿重及脏器系数明显增加（P＜0.01），并且具有一定的剂量-效应关系；4-NP和BPA高中低三种剂量都能够使子宫内膜上皮增高，平滑肌增厚，PCNA蛋白表达增强（P＜0.05）。低剂量的4-NP（50mg/kg）和BPA（200mg/kg）混合喂饲后上述指标均较单独喂饲组显著增加（P＜0.01）。（3）第一部分筛查出的EEDs暴露水平增高的性早熟患儿经中药治疗三个月后，子宫体积、血清E₂含量、骨矿含量、骨密度、血清骨钙素含量较治疗前明显减小或降低（P＜0.01）。4-NP和BPA染毒的实验动物经中药治疗后，所有治疗组的子宫湿重、脏器系数、骨密度、子宫内膜上皮高度、子宫平滑肌厚度及PCNA蛋白表达均比实验组明显降低（P＜0.05）。中药治疗方案能够显著下调ERα及ERβmRNA在子宫、乳腺和骨骺端的表达水平（P＜0.05），降低ERα及ERβ在子宫内膜上皮细胞、乳腺导管上皮细胞和骨骺端生长板软骨细胞的蛋白表达水平（P＜0.05）；能够显著下调EGFR mRNA在子宫、乳腺的表达水平（P＜0.05），降低EGFR在子宫内膜上皮细胞、乳腺导管上皮细胞的蛋白表达水平（P＜0.05）；能够显著下调IGF-1R mRNA在干骺端的表达水平（P＜0.05），降低IGF-1R在骨骺端生长板软骨细胞的蛋白表达水平（P＜0.05）。结论：（1）当前正常儿童已经较普遍地暴露于环境内分泌干扰物，只不过暴露程度比性早熟患儿略轻而已；EEDs暴露与儿童性早熟的发病有密切关系，是其重要的致病因素之一；不同种类的EEDs对靶器官的致病作用强度有所不同。（2）动物染毒实验进一步证实了EEDs的拟雌激素作用及其剂量-效应关系；进一步验证了临床研究结果及其推论，证实EEDs的暴露程度与性早熟发病有密切关系；不同种类EEDs之间可以发挥联合增效作用，暴露人群更易受到不良影响；为子宫营养试验提供了更为敏感的检测指标。（3）制定了针对EEDs拟雌激素作用的中药治疗方案；研究结果表明所用中药能够有效拮抗EEDs的雌激素样活性，明显减轻EEDs对机体性发育及骨骼发育造成的不良影响；能够显著下调靶细胞雌激素受体的表达水平，降低机体对EEDs作用的敏感性；能够显著下调靶细胞生长因子受体的表达水平，干扰交叉对话机制，进一步加强其对EEDs拟雌激素活性的拮抗作用。更多还原

【Abstract】 Objective: To explore the role of environmental endocrine disruptors（EEDs） in the onset of precocious puberty（PP） and the therapeutic effect of traditional Chinese medicine.Methods: （1） The blood samples were collected from 73 cases of precocious puberty patients and 38 cases of normal children. The concentrations of 4-nonylphenol（4-NP） , 1,1-dichloro-2,2,bis（p-chlorophenyl）ethylene（p,p’-DDE） and di-2-ethylhexyl phthalate（DEHP） in the blood serum samples were measured by using reversed-phase high performance liquid chromatography（HPLC）. The volume of uterus and ovary, bone density, and content of estradiol（E2） in the serum were determined at the same time. Contents of EEDs in the blood serums of precocious puberty cases and indices of the target organs were analyzed by correlation and regression methods. （2） The vehicle （peanut oil）, ethinyl estradiol（EE, 0.3mg/kg positive control） , 4-NP（50mg/kg,100mg/kg,200mg/kg）, bisphenol（BPA,200mg/kg,400mg/kg,600mg/kg）,DEHP（100mg/kg,250mg/kg,500mg/ kg） and a mixture of 4-NP 50mg/kg and BPA 200mg/kg were given orally（by gavage ） q.d. to immature female Sprague-Dawley rats on the 21^sd, 22^nd,23^rd postnatal days. The rats were sacrificed 24 hours after the last administration. Uterine wet weight, uterine/body weight ratio, height of the luminal epithelium, thickness of the myometrium and protein level of proliferating cell nuclear antigen（PCNA） of rat uterine were analysed by one-way ANOVA. （3） The therapeutic project of traditional Chinese medicine was established to treat PP children and the experimental animals. Developing parameters of reproductive organs and bone were observed. Using methods of real— time fluorescence quantitative PCR and immunohistochemistry, we detected the expression level of four receptors on target organs（uterine, breast, bone）, including estrogen receptor alpha（ERα） estrogen receptor beta（ERβ） epidermal growth factor receptor（EGFR） and insulin like factor-1 receptor（ IGF-1R）. The data were analysed by one-way ANOVA and T test.Results: （1） In the normal control group, p,p’-DDE was detected in all theblood samples（14.93 - 40.39ng/ml）, but 4-NP and DEHP were only detected in some of the samples（ND - 6.77 ng/ml,ND - 17.61 ng/ml）. Levels of 4-NP p,p’-DDE and DEHP in the serum of the precocious puberty group were notably higher than that of the control group （P < 0.01） .In the precocious puberty group, positive correlations were found between 4-NP and volume of uterus, volume of ovary and bone density (（r=0.394, 0.286,0.237; P < 0.01）; p,p’-DDE and volume of uterus also had a positive correlation(（r=0.306;P < 0.01）. The influence intensity of 4-NP was higher than that of p,p’-DDE. （2） In animal experiments, uterine wet weight and uterine/body weight ratio were significantly higher in the 4-NP 200mg/kg and 100mg/kg, BPA 600mg/kg and 400mg/kg, and mixture group（4-NP 50mg/kg and BPA 200mg/kg） （P < 0.05） . Height of luminal epithelium, thickness of myometrium,as well as protein level of PCNA of rat uterine were significantly increased in the three groups of 4-NP, the three groups of BPA, and the mixture group （P < 0.05） , and a dose-response relationship was observed. Levels of aboved parameters in the mixture group were significantly higher than that in the single dose groups （P < 0.05） . （3） Volume of uterus, content of estradiol（E2） in the serum and bone density, bone mineral content, content of osteocalcin（OST） in the serum were decreased in patients after treating with traditional Chinese medicine （P < 0.05） . Uterine wet weight, uterine/body weight ratio, height of the luminal epithelium, thickness of the myometrium, protein level of PCNA of rat uterine and bone density of rats were notably decreased in animals treated with Chinese traditional medicine （P < 0.05） . After the therapy, mRNA and protein levels of ER α and ER β were significantly down regulated in uterine, breast and bone（P < 0.05）. The mRNA and protein level of EGFR were significantly regulated down-ward in uterine and breast （P < 0.05） . The mRNA and protein level of IGF-1R were also significantly regulated down-ward in bones （P<0.05） .Conclusions: （1） Normal children and children with precocious puberty are all contaminated by EEDs, and the later are exposured to higher EEDs. There is a close relationship between EEDs and the onset of precocious puberty, and EEDs are important factors inducing the disease. Different kinds of EEDs have different influence intensity on target organs. （2） The estrogenic effects of EEDs and their dose-response relationship were validated by uterotrophic assay in immature SD rats. Different kinds of EEDs below no effect levels, if combined, could also evoke additive effects, exerting estrogenic activitiy. （3） Traditional Chinese medicine couldregulate and decrease the expression of ERα ,ERβ,EGFR and IGF-1R on the target organs. Through this mechanism, treatment with traditional Chinese medicine, the medicine could inhibit the estrogenic effects of EEDs and slow down the accelerated development of reproductive organs and skeleton in PP patients.更多还原