本文選題：內(nèi)分泌干擾物 + 性別發(fā)育�。� 參考：《大連理工大學(xué)》2015年博士論文

【摘要】：內(nèi)分泌干擾物(Endocrine disrupting chemicals, EDCs)廣泛存在于空氣、土壤、水體等環(huán)境介質(zhì)中,能夠干擾動(dòng)物性別發(fā)育過程及性別穩(wěn)定維持從而造成生物種群的性別比例失衡、生物個(gè)體性早熟、性反轉(zhuǎn)等性別發(fā)育異�，F(xiàn)象。最近研究發(fā)現(xiàn)在哺乳動(dòng)物的性別發(fā)育過程中,除性染色體上的關(guān)鍵基因,位于常染色體上一系列轉(zhuǎn)錄因子和信號(hào)分子的調(diào)控對(duì)雙潛能性腺的分化具有關(guān)鍵作用,性成熟后諸多性別決定基因也參與維系性腺的正常功能和性別穩(wěn)定過程。因此,基于常染色體上的性別決定基因研究EDCs對(duì)哺乳動(dòng)物性別發(fā)育過程和性別穩(wěn)定維持的基因調(diào)控通路的影響,對(duì)闡明其生殖和發(fā)育毒性機(jī)理具有重要意義。本論文以抗雌激素效應(yīng)化合物他莫昔芬(Tamoxifen, TAM)為模型化學(xué)物,研究其對(duì)小鼠性別發(fā)育和性別穩(wěn)定維持過程常染色體上關(guān)鍵調(diào)控基因的影響,并基于這一研究結(jié)果進(jìn)一步選擇兩種典型環(huán)境內(nèi)分泌干擾物—雙酚A(Bisphenol A, BPA)和三丁基錫(Tributyltin, TBT),探討其干擾小鼠性別發(fā)育過程的分子調(diào)控機(jī)理。本論文的主要研究內(nèi)容和結(jié)果如下：1.胚胎期暴露TAM對(duì)小鼠性別決定基因的影響。ICR孕鼠在妊娠第10.5天(days post-coitum, dpc)腹腔注射1 mg/kg-bw TAM,13.5 dpc取胎鼠生殖嵴,采用實(shí)時(shí)反轉(zhuǎn)錄多聚酶鏈?zhǔn)椒磻?yīng)(Realtime reverse transcription polymerase chain reaction, Realtime RT-PCR)、整胚原位雜交對(duì)性別決定基因表達(dá)進(jìn)行檢測(cè)。結(jié)果發(fā)現(xiàn)性別決定關(guān)鍵期暴露TAM能夠誘導(dǎo)雌性生殖嵴中血小板源生長因子受體-a(Platelet-derived growth factor receptor-a, Pdgfra)過表達(dá)且表達(dá)模式與雄性生殖嵴類似,表明TAM能夠誘導(dǎo)睪丸特異性體腔血管生成。同時(shí)TAM顯著上調(diào)小鼠雌性生殖嵴中睪丸特異性轉(zhuǎn)錄因子Sry相關(guān)基因9(SRY-related HMG box gene 9, Sox9)表達(dá),但下調(diào)卵巢決定關(guān)鍵因子—叉頭狀轉(zhuǎn)錄因子基因2(Forkhead/winged helix transcription factor gene 2, Foxl2)表達(dá)。SOX9和FOXL2蛋白表達(dá)水平的變化和基因表達(dá)變化一致。小鼠胚胎生殖嵴體外培養(yǎng)實(shí)驗(yàn)進(jìn)一步證實(shí)TAM能夠顯著上調(diào)雌性生殖嵴中Pdgfra和成纖維細(xì)胞生長因子9(Fibroblast growth factor-9, Fgf9)表達(dá),并降低雄性生殖嵴中Fgf9的表達(dá)。雌二醇和睪酮的比值(E2/T)在TAM暴露組中也顯著降低。結(jié)果提示TAM抑制小鼠雌性生殖嵴常染色體上雌性特異性關(guān)鍵基因表達(dá),激活雄性決定基因的表達(dá),干擾性別發(fā)育過程,從而異常起始雌性生殖嵴中睪丸發(fā)育調(diào)控通路。2.性成熟期TAM暴露對(duì)雌性小鼠性別穩(wěn)定調(diào)控通路關(guān)鍵基因的影響。向性成熟的ICR雌性小鼠,連續(xù)7d分別腹腔注射75或225 mg/kg-bw TAM。暴露結(jié)束5d后,收集小鼠血清和卵巢,采用Realtime RT-PCR、免疫組織化學(xué)、蛋白免疫印跡(Western blot)對(duì)性別決定基因和蛋白的表達(dá)和分布進(jìn)行檢測(cè)。結(jié)果發(fā)現(xiàn)TAM上調(diào)了卵巢中睪丸索成分血小板內(nèi)皮細(xì)胞粘附分子-1 (Platelet/endothelial cell adhesion molecule 1, PECAM-1)表達(dá),表明TAM誘導(dǎo)卵巢產(chǎn)生睪丸特異性血管內(nèi)皮細(xì)胞。此外,TAM還能夠上調(diào)Sox9和Fgf9基因表達(dá)；抑制Foxl2.無翼乳房腫瘤病毒相關(guān)整合位點(diǎn)4(Wingless-related MMTV integration site 4, Wnt4)和其下游基因卵泡抑制素(Follistatin, Fst)的表達(dá)。TAM暴露導(dǎo)致激素水平紊亂,雌二醇、孕酮、促卵泡生成素和黃體生成素水平降低,而睪酮含量增加。結(jié)果表明性成熟期TAM暴露可以抑制卵巢維持因子Foxtt和Wnt4及其下游基因的表達(dá),而激活睪丸決定因子Sox9和Fgf9,啟動(dòng)Sox9和Fgf9之間的反饋調(diào)節(jié)通路,從而干擾性成熟雌性小鼠卵巢功能維持和性別穩(wěn)定。3.胚胎期和哺乳期暴露BPA對(duì)雄性仔鼠性別發(fā)育調(diào)控通路關(guān)鍵基因的影響。ICR小鼠孕鼠從0.5 dpc直至出生后21d(Postnatal day, PND21)連續(xù)暴露0.1 mg/kg-bw BPA。取PND0、7、28、49雄性仔鼠,收集血清及睪丸組織。采用Realtime RT-PCR、Western blot和免疫組織化學(xué)等方法對(duì)參與性別發(fā)育過程的關(guān)鍵基因和蛋白進(jìn)行檢測(cè)。結(jié)果發(fā)現(xiàn),BPA未顯著影響雄性仔鼠性成熟期FGF9及SOX9蛋白的表達(dá),但抑制出生早期和性成熟期抗繆勒氏管激素(Anti-Mullerian hormone, Amh)、Sox9和Fgf9表達(dá)。同時(shí)BPA暴露下調(diào)了雄性仔鼠各時(shí)期鋅指轉(zhuǎn)錄因子3ATA結(jié)合蛋白4(Zinc finger transcription factor GATA-binding protein 4, Gata4)的表達(dá),使其在青春期未正常表達(dá)上調(diào)。此外,BPA誘導(dǎo)Foxl2、Wnt4、Fst和X染色體DSS-AHC決定區(qū)基因1(DSS-AHC critical region on the X chromosome gene 1, Dea1)表達(dá)上調(diào)。結(jié)果提示發(fā)育早期BPA暴露可抑制雄性仔鼠睪丸調(diào)控通路,激活卵巢調(diào)控通路,揭示了BPA誘導(dǎo)雄性動(dòng)物雌性化的潛在機(jī)制。4.胚胎期和哺乳期暴露TBT對(duì)雄性仔鼠性別發(fā)育調(diào)控通路關(guān)鍵基因的影響。ICR小鼠孕鼠從0.5 dpc直至PND21連續(xù)暴露20μg/kg-bw TBT。仔鼠出生后,在PND0、7、28、49取雄性小鼠血清及睪丸組織。采用Realtime RT-PCR、Western blot和免疫組織化學(xué)等方法對(duì)參與性別發(fā)育的關(guān)鍵基因和蛋白進(jìn)行檢測(cè)。結(jié)果發(fā)現(xiàn)胚胎期暴露TBT能夠增加成年雄性仔鼠血清中E2/T,而對(duì)雌性仔鼠血清中E2/T無影響。TBT顯著抑制PND0時(shí)Sox9、Fgf9和Amh基因表達(dá)水平,隨時(shí)間延長無顯著影響。TBT改變了生精細(xì)胞與精子形成相關(guān)基因Gata4的表達(dá)趨勢(shì),導(dǎo)致其表達(dá)異常升高并誘導(dǎo)其過早表達(dá)。TBT暴露未顯著改變雄性仔鼠睪丸中卵巢發(fā)育特異性基因Foxl2等表達(dá)水平和時(shí)間規(guī)律。結(jié)果表明胚胎期和哺乳期暴露TBT干擾子代雄性性別發(fā)育過程,至成年期性激素分泌仍呈紊亂狀態(tài)。本論文研究了EDCs中三種代表性化學(xué)物對(duì)小鼠常染色體上性別決定基因的影響,發(fā)現(xiàn)基因調(diào)控通路的紊亂和其造成的性別發(fā)育和性別穩(wěn)定異常一致,揭示了EDCs新的生殖發(fā)育毒性作用的潛在機(jī)制。該結(jié)果有助于闡明環(huán)境內(nèi)分泌干擾物暴露與哺乳動(dòng)物及人類的性別比例異常和生殖障礙之間的關(guān)聯(lián),同時(shí)有助于揭示發(fā)育早期接觸EDCs對(duì)機(jī)體功能發(fā)育及后期健康的長期影響機(jī)制,為EDCs的健康風(fēng)險(xiǎn)評(píng)價(jià)提供科學(xué)依據(jù)。
[Abstract]:Endocrine disrupting interferons (Endocrine disrupting chemicals, EDCs) are widely existed in air, soil, water and other environmental mediums, which can interfere with the sex development of animals and maintain sex stability, resulting in the gender imbalance of biological population, individual precocious precocity, sex reversal and other sex dysplasia. Recent studies have found that breastfeeding In the process of sex development in animals, the key genes on the sex chromosome, the regulation of a series of transcription factors and signal molecules on the autosomes play a key role in the differentiation of the dual potential gonads. After sexual maturation, many sex determination genes are also involved in the normal function and gender stability of the gonads. Therefore, the autosomal based autosomes are on the autosomes. The effect of EDCs on the sex development process and the gene regulation pathway of gender stability in mammals is of great significance to elucidate the mechanism of reproductive and developmental toxicity. This paper uses the anti estrogen effect compound tamoxifen (Tamoxifen, TAM) as a model chemical, and studies its sex development and sex in mice. The key regulatory genes on the autosomes in the stable maintenance process, and based on the results of this study, we further selected two typical environmental endocrine disruptors - bisphenol A (Bisphenol A, BPA) and three butyl tin (Tributyltin, TBT) to discuss the molecular regulation mechanism that interfered with the sex development of mice. The results are as follows: 1. the effects of exposure to TAM on the sex determination genes of mice in the embryo period.ICR mice were intraperitoneally injected with 1 mg/kg-bw TAM (days post-coitum, DPC) on day 10.5 of pregnancy, and the reproductive crista of fetal mice was taken by 13.5 dpc, and the real time reverse transcription polymerase chain reaction (Realtime reverse transcription polymerase) was used as a whole embryo. The expression of sex determined gene was detected by in situ hybridization. The results showed that exposure to TAM in the critical stage of sex determination could induce the overexpression of -a (Platelet-derived growth factor receptor-A, Pdgfra) in female reproductive crista, and the expression pattern was similar to that of male reproductive crista, indicating that TAM can induce testicular specific body cavity. At the same time, TAM significantly up-regulated the expression of Sry related gene 9 (SRY-related HMG box gene 9, Sox9) in the female reproductive crista of mice, but down regulated the key factor of the ovarian decision - the fork head transcription factor gene 2 (Forkhead/winged helix transcription factor gene 2) and the expression of water In vitro culture experiment of mouse embryo reproductive crista further confirmed that TAM could significantly increase the expression of Pdgfra and fibroblast growth factor 9 (Fibroblast growth factor-9, Fgf9) in female reproductive crista, and reduce the expression of Fgf9 in the male reproductive crista. The ratio of estradiol and testosterone (E2/T) in TAM exposure The results suggest that TAM inhibits the expression of key female specific key genes on the autosomes of female reproductive crista, activates the expression of the male determining genes and interferes with the process of sex development, so that the abnormal initiation of the testicular development regulation pathway in the female reproductive crista in the female reproductive crista is to regulate the sexual stability of female mice by TAM exposure in the.2. sexual maturity stage. The effect of bond gene. The expression and distribution of sex determined genes and proteins were detected by Realtime RT-PCR, immunohistochemical staining and protein immunoblotting (Western blot) in ICR female mice of mature ICR, after continuous 7d intraperitoneal injection of 75 or 225 mg/kg-bw TAM. exposures to 5D, and the expression and distribution of sex determined genes and proteins were detected. The expression of the platelet endothelial cell adhesion molecule -1 (Platelet/endothelial cell adhesion molecule 1, PECAM-1) in the ovary of the ovary showed that TAM induced the ovary to produce testicular specific vascular endothelial cells. In addition, TAM could also up regulate the expression of Sox9 and Fgf9 genes, and inhibit the Foxl2. pterygoid tumor virus related integration site 4 (Wingl). Ess-related MMTV integration site 4, Wnt4) and its downstream gene follicle inhibin (Follistatin, Fst) expression.TAM exposure leads to hormone levels disorder, estradiol, progesterone, follicle stimulating hormone and luteinizing hormone levels decrease, and testosterone levels are increased. The results show that TAM exposure at sexual maturity can inhibit ovarian maintenance factor Foxtt and Wnt. The expression of 4 and its downstream genes, while activating the testicular determinants Sox9 and Fgf9, and starting the feedback regulation pathway between Sox9 and Fgf9, thus interfering with the ovarian function maintenance of the mature female mice and the gender stable.3. embryo and the lactating stage, the effect of BPA on the key genes of sex development of male offspring in male mice.ICR mice from 0.5 DPC. 21d (Postnatal day, PND21) was continuously exposed to 0.1 mg/kg-bw BPA. to collect PND0,7,28,49 male offspring and collect serum and testicular tissues. The key genes and proteins involved in sex development process were detected by Realtime RT-PCR, Western blot and immunohistochemistry. The results showed that BPA did not significantly affect male offspring sex. The expression of FGF9 and SOX9 protein at mature stage, but inhibit the expression of Anti-Mullerian hormone, Amh, Sox9 and Fgf9 in the early stage of birth and sexual maturity, while BPA exposure down regulated the expression of 3ATA binding protein 4 of the zinc finger transcription factor of the male offspring (Zinc finger transcription). In addition, BPA induced Foxl2, Wnt4, Fst and X chromosome DSS-AHC determinant gene 1 (DSS-AHC critical region on the X) expression up-regulated. The results suggest that early developmental exposure inhibits the regulation of testicular regulation in male offspring, activates the ovarian regulation pathway, and reveals the induced male movement. The potential mechanism of physical feminization.4. embryo and lactation stage exposure TBT effect on the key genes of sex development control pathway in male offspring mouse.ICR mice pregnant mice from 0.5 DPC until PND21 continuous exposure to 20 g/kg-bw TBT. offspring were born in PND0,7,28,49 male mice serum and testicular tissue. Realtime RT-PCR, Western blot and immune group were used. The detection of key genes and proteins involved in sex development was detected by weave chemistry. The results showed that exposure to TBT in embryo stage could increase the E2/T in the serum of adult male offspring, but there was no effect of.TBT on the expression level of Sox9, Fgf9 and Amh in the serum of female offspring, and no significant influence of.TBT on the E2/T. The expression trend of cell and sperm formation related gene Gata4, resulting in abnormal expression of its expression and induction of premature expression of.TBT exposure did not significantly change the expression level and time regularity of the ovarian developmental specific gene Foxl2 in the testicles of male offspring. The results showed that the embryo and lactation period exposed TBT interfering the male sex development process to adulthood. The effect of three representative chemicals on the sex determination genes on the autosomes of EDCs was studied in this paper. It was found that the disorder of the gene regulation pathway was unusually consistent with the sex development and gender stability, which revealed the potential mechanism of EDCs's new reproductive toxicity. It helps to elucidate the relationship between environmental endocrine disruptors exposure and the sexual abnormality of sex and reproductive disorders in mammals and humans, and helps to reveal the long-term mechanism of the impact of early exposure to EDCs on the functional development and later health of the body, and provides a scientific basis for the assessment of the health risk of EDCs.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R114

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