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  本文選題：環(huán)境內(nèi)分泌干擾物　切入點：性早熟　出處：《復旦大學》2012年碩士論文　論文類型：學位論文

【摘要】：目的：(1)建立靈長類—爪哇猴由環(huán)境內(nèi)分泌干擾物(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驗證滋陰瀉火中藥對EEDs擬雌激素活性的拮抗作用,以更準確地模擬人類的疾病過程及藥物的治療作用。(3)采用分子生物學實驗方法,研究并闡明中藥拮抗EEDs擬雌激素活性的作用機制,為開發(fā)具有自主知識產(chǎn)權的有效拮抗EEDs生殖毒性的中藥制劑提供更為恰當?shù)睦碚撘罁?jù)。 方法：(1)預實驗：以青春前期雌性爪哇猴為實驗對象,選取具有代表性的EEDs—壬基酚(4-nonylphenol,4-NP)及雙酚A (bisphenol A,BPA)作為染毒物質,復制爪哇猴性早熟的疾病模型。爪哇猴分為兩個月齡組,每個年齡組分高劑量染毒組、低劑量染毒組及對照組。每日空腹給藥。以陰道脫落細胞成熟指數(shù)、子宮容積、子宮濕重、臟器系數(shù)、子宮內(nèi)膜厚度、子宮肌層厚度、子宮內(nèi)膜上皮細胞高度、子宮腺上皮高度為指標。結果采用單因素方差分析,觀察EEDs的擬雌激素作用。確定爪哇猴性早熟疾病模型建立的最佳月齡、最佳染毒劑量及最佳染毒時間。(2)以最佳月齡的青春前期雌性爪哇猴為實驗對象,以最佳劑量的壬基酚(nonylphenol,4-NP)及雙酚A (bisphenol A,BPA)復制聯(lián)合染毒的性早熟疾病模型。爪哇猴隨機分為染毒組,治療組及對照組,每日空腹給藥。染毒組喂飼4-NP及BPA,治療組喂飼4-NP、BPA及中藥,對照組喂飼溶劑玉米油。以雌激素水平、陰道脫落細胞成熟指數(shù)、子宮濕重、臟器系數(shù)、子宮內(nèi)膜厚度、子宮肌層厚度、子宮內(nèi)膜上皮細胞高度、子宮腺上皮高度為指標,結果采用單因素方差分析,驗證中藥對EEDs擬雌激素活性的拮抗作用。(3)采用實時熒光定量聚合酶鏈式反應(RT-PCR)及免疫印跡方法(Western Blotting)方法,檢測雌激素受體α (estrogen receptor α, ERa)、雌激素受體β (estrogen receptor β, ERβ)、表皮生長因子受體(epidermal growth factor receptor, EGFR)、胰島素樣生長因子1受體(insulin-like growth factor-1receptor, IGF-1R)、膽固醇側鏈裂解酶(cytochrome P450,family ll,subfamily A,polypeptide1, CYP11A1)、芳香化酶(cytochrome P450,family19,subfamily A,polypeptide1, CYP19A1)基因及蛋白表達水平。結果采用單因素方差分析。通過染毒組、治療組及對照組的相互對比,闡明中藥拮抗EEDs擬雌激素活性的作用機制。 結果：(1)確定爪哇猴由EEDs引致性早熟疾病模型,應以26月齡為最佳月齡,以低劑量BPA (200mg/kg)及4-NP (50mg/kg)為最佳染毒劑量,以4周為最佳染毒時間。(2)染毒組與對照組比較雌激素水平明顯升高,陰道脫落細胞成熟指數(shù)顯著增加,子宮濕重、臟器系數(shù)、子宮內(nèi)膜上皮細胞高度、子宮腺上皮高度顯著增加,子宮內(nèi)膜及肌層厚度明顯增高。治療組與染毒組相比,上述指標均顯著降低(p0.05)。(3) RT-PCR方法檢測顯示BPA.4-NP聯(lián)合染毒可導致ERα、 ERP、EGFR、IGF-1R、CYP11A1、CYP19A1的mRNA表達水平顯著上調(p0.05)。而中藥干預后,可使上述基因表達顯著下調(p0.05)。(4)經(jīng)Western Blot方法驗證BPA、4-NP聯(lián)合染毒可導致ERα、ERβ、EGFR、CYP19A1的蛋白表達水平顯著上調(p0.05)。而中藥干預后,可使上述蛋白表達顯著下調(p0.05)。 結論：(1)我們成功地建立了靈長類——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驗證了滋陰瀉火中藥對EEDs的擬雌激素活性具有顯著的拮抗作用；(3)滋陰瀉火中藥可通過使生殖器官ER、EGFR、IGFR表達的下調以及雌激素合成關鍵酶的表達下調,多水平、多靶點地發(fā)揮其對EEDs擬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.

【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R725.8;R-332

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