爪哇猴由环境内分泌干扰物引致性早熟疾病模型的建立及中药作用机制的研究

发布时间：2018-03-03 12:19

本文选题：环境内分泌干扰物　切入点：性早熟　出处：《复旦大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：(1)建立灵长类—爪哇猴由环境内分泌干扰物(environmental endocrine disruptors, EEDs)引致的性早熟疾病模型。(2)在爪哇猴性早熟模型上验证滋阴泻火中药对EEDs拟雌激素活性的拮抗作用,以更准确地模拟人类的疾病过程及药物的治疗作用。(3)采用分子生物学实验方法,研究并阐明中药拮抗EEDs拟雌激素活性的作用机制,为开发具有自主知识产权的有效拮抗EEDs生殖毒性的中药制剂提供更为恰当的理论依据。方法：(1)预实验：以青春前期雌性爪哇猴为实验对象,选取具有代表性的EEDs—壬基酚(4-nonylphenol,4-NP)及双酚A (bisphenol A,BPA)作为染毒物质,复制爪哇猴性早熟的疾病模型。爪哇猴分为两个月龄组,每个年龄组分高剂量染毒组、低剂量染毒组及对照组。每日空腹给药。以阴道脱落细胞成熟指数、子宫容积、子宫湿重、脏器系数、子宫内膜厚度、子宫肌层厚度、子宫内膜上皮细胞高度、子宫腺上皮高度为指标。结果采用单因素方差分析,观察EEDs的拟雌激素作用。确定爪哇猴性早熟疾病模型建立的最佳月龄、最佳染毒剂量及最佳染毒时间。(2)以最佳月龄的青春前期雌性爪哇猴为实验对象,以最佳剂量的壬基酚(nonylphenol,4-NP)及双酚A (bisphenol A,BPA)复制联合染毒的性早熟疾病模型。爪哇猴随机分为染毒组,治疗组及对照组,每日空腹给药。染毒组喂饲4-NP及BPA,治疗组喂饲4-NP、BPA及中药,对照组喂饲溶剂玉米油。以雌激素水平、阴道脱落细胞成熟指数、子宫湿重、脏器系数、子宫内膜厚度、子宫肌层厚度、子宫内膜上皮细胞高度、子宫腺上皮高度为指标,结果采用单因素方差分析,验证中药对EEDs拟雌激素活性的拮抗作用。(3)采用实时荧光定量聚合酶链式反应(RT-PCR)及免疫印迹方法(Western Blotting)方法,检测雌激素受体α (estrogen receptor α, ERa)、雌激素受体β (estrogen receptor β, ERβ)、表皮生长因子受体(epidermal growth factor receptor, EGFR)、胰岛素样生长因子1受体(insulin-like growth factor-1receptor, IGF-1R)、胆固醇侧链裂解酶(cytochrome P450,family ll,subfamily A,polypeptide1, CYP11A1)、芳香化酶(cytochrome P450,family19,subfamily A,polypeptide1, CYP19A1)基因及蛋白表达水平。结果采用单因素方差分析。通过染毒组、治疗组及对照组的相互对比,阐明中药拮抗EEDs拟雌激素活性的作用机制。结果：(1)确定爪哇猴由EEDs引致性早熟疾病模型,应以26月龄为最佳月龄,以低剂量BPA (200mg/kg)及4-NP (50mg/kg)为最佳染毒剂量,以4周为最佳染毒时间。(2)染毒组与对照组比较雌激素水平明显升高,阴道脱落细胞成熟指数显著增加,子宫湿重、脏器系数、子宫内膜上皮细胞高度、子宫腺上皮高度显著增加,子宫内膜及肌层厚度明显增高。治疗组与染毒组相比,上述指标均显著降低(p0.05)。(3) RT-PCR方法检测显示BPA.4-NP联合染毒可导致ERα、 ERP、EGFR、IGF-1R、CYP11A1、CYP19A1的mRNA表达水平显著上调(p0.05)。而中药干预后,可使上述基因表达显著下调(p0.05)。(4)经Western Blot方法验证BPA、4-NP联合染毒可导致ERα、ERβ、EGFR、CYP19A1的蛋白表达水平显著上调(p0.05)。而中药干预后,可使上述蛋白表达显著下调(p0.05)。结论：(1)我们成功地建立了灵长类——爪哇猴由EEDs引致的性早熟疾病模型；(2)在爪哇猴性早熟模型上验证了滋阴泻火中药对EEDs的拟雌激素活性具有显著的拮抗作用；(3)滋阴泻火中药可通过使生殖器官ER、EGFR、IGFR表达的下调以及雌激素合成关键酶的表达下调,多水平、多靶点地发挥其对EEDs拟雌激素活性及生殖毒性的拮抗作用。
[Abstract]:Objective: (1) the establishment of primate - Java monkey consists of environmental endocrine disruptors (environmental endocrine, disruptors, EEDs) model induced precocious disease. (2) verification of zyxhr on antagonism of EEDs estrogenic activity in Java monkey precocious puberty model, the therapeutic effect of a more accurate simulation of the disease process and drug human. (3) using molecular biology experimental methods, research and mechanism of traditional Chinese medicine EEDs antagonistic estrogenic activity, traditional Chinese medicine is developed with independent intellectual property rights effectively antagonize the reproductive toxicity of EEDs provide the appropriate theoretical basis.
Methods: (1) pre experiment: in prepubertal female monkey Java as the experimental object, selects the representative EEDs (4-nonylphenol, 4-NP) - nonylphenol and bisphenol A (bisphenol A BPA) as poisonous substances, Java monkey precocious disease replication model. Java monkey is divided into two age groups, each the age group of high dose group, low dose group and control group. Fasting daily dosing. With the maturation index, uterine volume, Komiya Shige, organ coefficient, endometrial thickness, uterine muscle thickness, endometrial epithelial cell height, glandular epithelium height index. Results with single factor analysis the variance, estrogenic effects of EEDs. To determine the best months to establish the model of Java monkey precocious disease, the optimal dose and the best exposure time. (2) to the best month old prepubertal female monkey Java as the experimental object, with the best agent The amount of nonylphenol (nonylphenol, 4-NP) and bisphenol A (bisphenol A BPA) model combined exposure of precocious disease. Java monkeys were randomly divided into control group, treatment group and control group, fasting daily dosing. Exposed groups were fed with 4-NP and BPA, the treatment group fed with 4-NP, BPA and traditional Chinese Medicine, control group fed corn oil by solvent. The estrogen level, maturation index, Komiya Shige, organ coefficient, endometrial thickness, uterine muscle thickness, the height of endometrial epithelium, glandular epithelium height index, the single factor variance analysis, verification of traditional Chinese medicine on antagonism of EEDs estrogenic activity. (3) using real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and Western blot (Western Blotting) method, the detection of estrogen receptor alpha (estrogen alpha, receptor, ERa), estrogen receptor beta (estrogen beta receptor and ER beta), epidermal growth factor Receptor (epidermal growth factor receptor, EGFR), insulin-like growth factor 1 receptor (insulin-like growth, factor-1receptor, IGF-1R), cholesterol side chain cleavage (cytochrome P450, family ll, subfamily A, polypeptide1, CYP11A1), aromatase (cytochrome P450, family19 subfamily, A, polypeptide1, CYP19A1) level of gene and protein expression. Results the single factor analysis of variance. By contrast control group, treatment group and control group, to clarify the mechanism of Chinese herbal medicine EEDs antagonistic estrogenic activity.
Results: (1) determined by EEDs induced premature Java monkey disease model, with 26 month old is the best month of age, with a low dose of BPA (200mg/kg) and 4-NP (50mg/kg) is the best dose for 4 weeks, the best exposure time. (2) the exposure group compared with the control group significantly increased levels of estrogen, vaginal exfoliated cells maturity index increased significantly, uterine wet weight, organ coefficient, height of endometrial epithelium, glandular epithelium height increased significantly, endometrial and myometrial thickness increased significantly. The treatment group compared with the control group, the above indexes were significantly lower (P0.05). (3) RT-PCR assay showed that BPA.4-NP plus exposure cause ER alpha, ERP, EGFR, IGF-1R, CYP11A1, CYP19A1 mRNA expression level was increased (P0.05). And the traditional Chinese medicine intervention, the gene expression was significantly reduced (P0.05). (4) by Western Blot method to verify the BPA, 4-NP combined exposure can cause ER alpha, ER beta, EGF The protein expression level of R, CYP19A1 was significantly up-regulated (P0.05), and the expression of the above protein could be reduced significantly (P0.05) after the prognosis of Chinese medicine.
Conclusion: (1) we successfully established primates - Java EEDs induced precocious monkey by disease model; (2) in Java monkey precocious puberty model verified zyxhr on EEDs estrogenic activity inhibited; (3) zyxhr through the reproductive organs ER, EGFR, IGFR expression, and down-regulation of the expression of the key enzyme for synthesis of estrogen levels, multiple targets play an important role in the antagonism of EEDs to activity and reproductive toxicity of estrogen.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.8;R-332

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