【摘要】：多溴聯(lián)苯醚(Polybrominated Diphenyl Ethers, PBDEs)是新型持久性有機(jī)污染物。自Main KM等2007年首次報(bào)道隱睪癥患兒的患病率與母乳中PBDEs濃度存在關(guān)聯(lián)后,針對PBDEs的生殖毒性成為眾多學(xué)者關(guān)注的研究熱點(diǎn)。 目前,關(guān)于PBDEs暴露與人類生殖發(fā)育之間關(guān)系的人群研究資料非常有限。有關(guān)PBDEs生殖毒性的動(dòng)物試驗(yàn)雖然不斷增多,但針對青春期暴露于PBDEs對雄性生殖系統(tǒng)影響的系統(tǒng)報(bào)道較少,且所用染毒劑量遠(yuǎn)高于實(shí)際生活中人群的暴露水平,缺乏對應(yīng)于人群暴露水平的低劑量毒性研究資料。 針對上述研究不足,本研究首先通過山東某濱海開發(fā)區(qū)建立的出生隊(duì)列,就新生兒PBDEs暴露水平及其影響開展前瞻性研究；然后以人群暴露水平作為劑量參考,建立低劑量染毒動(dòng)物模型,系統(tǒng)觀察青春期前后低劑量BDE-209暴露對雄性生殖系統(tǒng)發(fā)育,特別是精子發(fā)育的影響；最后通過建立睪丸生殖細(xì)胞體外培養(yǎng)體系,探索PBDEs影響生殖健康的可能機(jī)制。 第一部分某地新生兒多溴聯(lián)苯醚暴露水平調(diào)查及其生殖發(fā)育毒性的研究 目的：探討新生兒體內(nèi)PBDEs暴露水平,分析其暴露與新生兒宮內(nèi)妊娠結(jié)局的相關(guān)性。 方法：選擇山東省某濱海開發(fā)區(qū)孕婦作為研究對象,追蹤隨訪孕婦妊娠過程及妊娠結(jié)局,同時(shí)對新生兒臍帶血血清PBDEs水平進(jìn)行測定,并對兩者關(guān)系進(jìn)行相關(guān)分析。 結(jié)果： 新生兒所有血清樣本(n=176)均能檢測到一種或多種PBDEs同系物。新生兒臍帶血清BDE-100(r=0.607, P0.01)、BDE-47(r=0.352, P0.05)、 BDE-99(r=0.412,P0.05)水平與其母親既往懷孕期間因胎兒不正常選擇人工流產(chǎn)呈現(xiàn)明顯的正相關(guān)。新生兒血清BDE-153水平與新生兒身長呈顯著正相關(guān)(r=0.452,P0.01)。 結(jié)論：山東某濱海開發(fā)區(qū)新生兒體內(nèi)普遍存在PBDEs暴露,新生兒體內(nèi)PBDEs水平與其宮內(nèi)不良妊娠發(fā)育結(jié)局存在一定的關(guān)聯(lián)。 第二部分雄性大鼠青春期前后十溴聯(lián)苯醚暴露的生殖發(fā)育毒性研究 目的：探討青春期前后BDE-209暴露對雄性大鼠性發(fā)育、生殖器腺發(fā)育、精子發(fā)育及生殖內(nèi)分泌功能的影響。 方法：24只SD雄性大鼠(4周齡)隨機(jī)分為4組,以不同濃度BDE-209(0、2、10、50mg/kg.d)灌胃染毒,1次/d,連續(xù)染毒一個(gè)月。染毒結(jié)束后,采集樣本并分析。 結(jié)果： 1、與對照組相比,50mg/kg BDE-209染毒組睪丸抗氧化酶SOD、GSH-Px以及標(biāo)記酶AKP活性均較對照組明顯下降(P0.05),睪丸病理切片可見空泡樣變。但各染毒組在體重、肛殖距、陰莖包皮分離時(shí)間以及各臟器系數(shù)等方面未見明顯改變。 2、BDE-209染毒后,精子活力呈現(xiàn)出隨BDE-209濃度增高而明顯下降的趨勢(P0.05),其中50mg/kg組精子活力下降最為明顯(P0.05)。BDE-209染毒組精子運(yùn)動(dòng)速率、前進(jìn)運(yùn)動(dòng)精子率較對照組也呈現(xiàn)出下降趨勢,其中50mg/kg組精子運(yùn)動(dòng)速率下降具有統(tǒng)計(jì)學(xué)意義(P0.05)。與此同時(shí),BDE-209染毒組的精子畸形率較對照組明顯增高(P0.05)。但未見BDE-209暴露對精子密度產(chǎn)生顯著性改變。 3、BDE-209染毒后,血清睪酮、血清雌二醇、睪丸組織睪酮含量與對照組相比,均未見顯著性改變(P0.05)。 結(jié)論：在本實(shí)驗(yàn)設(shè)計(jì)劑量下,青春期前后BDE-209暴露對大鼠睪丸的形態(tài)功能已產(chǎn)生一定的改變,尤其是對精子分化成熟過程產(chǎn)生了明顯影響,引起精子畸形率增加,精子活力下降,精子運(yùn)動(dòng)速率降低。 第三部分體外培養(yǎng)睪丸支持細(xì)胞暴露于十溴聯(lián)苯醚的毒性研究 目的：探討B(tài)DE-209對睪丸支持細(xì)胞的毒性作用。 方法：選用18天齡SD大鼠,取睪丸支持細(xì)胞進(jìn)行原代培養(yǎng),給予BDE-209染毒24h,采用MTT法測定細(xì)胞濃度,同時(shí)利用Annexin V-FITC試劑盒檢測支持細(xì)胞的凋亡情況。 結(jié)果： 1、大鼠睪丸支持細(xì)胞在培養(yǎng)36h后進(jìn)入對數(shù)增長期,48-72h是適宜的染毒窗口期。不同濃度BDE-209染毒24h后,隨染毒濃度增加,細(xì)胞存活率下降,其中當(dāng)BDE-209濃度≥30μg/ml時(shí),睪丸支持細(xì)胞的相對存活率較對照組明顯降低(P0.05)。 2、與對照組相比,BDE-209染毒組支持細(xì)胞T-AOC、SOD呈現(xiàn)下降趨勢,其中10、20μg/ml染毒組T-AOC、SOD活性較對照組明顯下降(P0.05)。 3、BDE-209染毒24h后,睪丸支持細(xì)胞的凋亡率出現(xiàn)增高趨勢,當(dāng)BDE-209濃度≥20μg/ml時(shí),睪丸支持細(xì)胞凋亡率較對照組顯著增高(P0.05)。 結(jié)論：BDE-209對體外培養(yǎng)的睪丸支持細(xì)胞具有細(xì)胞毒性作用,BDE-209暴露可引起睪丸支持細(xì)胞出現(xiàn)凋亡。
[Abstract]:Polybrominated Diphenyl Ethers (PBDEs) is a new type of persistent organic pollutant. Since the incidence of cryptorchidism in children was first reported by Main KM in 2007, the reproductive toxicity of PBDEs in breast milk has become a research hotspot.
At present, there is very limited data on the relationship between PBDEs exposure and human reproductive development. Animal trials on the reproductive toxicity of PBDEs are increasing, but systematic reports on the effects of pubertal exposure to PBDEs on the male reproductive system are scarce, and the dose used is much higher than the exposure level in real life. Lack of data for low dose toxicity studies corresponding to population exposure levels.
In view of the insufficiency of the above studies, this study firstly carried out a prospective study on the level of PBDEs exposure and its effects in newborns through a birth cohort in a coastal development area of Shandong Province, and then established an animal model of low-dose exposure to BDE-209 before and after puberty, taking the level of population exposure as a dose reference, to systematically observe the effects of low-dose BDE-209 exposure on male puberty. The effects of PBDEs on reproductive system development, especially sperm development, and the possible mechanism of PBDEs on reproductive health were explored by establishing an in vitro culture system of testicular germ cells.
The first part is a survey of the exposure level of polybrominated diphenyl ethers in a newborn and its reproductive toxicity.
Objective: To investigate the level of PBDEs exposure in neonates and analyze the correlation between PBDEs exposure and intrauterine pregnancy outcome.
Methods: The pregnant women in a coastal development area of Shandong Province were selected as the research object. The pregnancy process and pregnancy outcome were followed up. The serum PBDEs levels of umbilical cord blood were measured and the relationship between them was analyzed.
Result:
One or more PBDEs homologues were detected in all neonatal serum samples (n = 176). The levels of BDE-100 (r = 0.607, P 0.01), BDE-47 (r = 0.352, P 0.05), BDE-99 (r = 0.412, P 0.05) in neonatal umbilical cord serum were positively correlated with maternal abnormal selection of induced abortion during previous pregnancy. Neonatal length was positively correlated (r=0.452, P0.01).
Conclusion: PBDEs are ubiquitous in newborns of a coastal development zone in Shandong Province. The level of PBDEs in newborns is related to the adverse pregnancy outcomes.
The second part is about the reproductive and developmental toxicity of ten brominated diphenyl ethers before and after puberty in male rats.
Objective: To investigate the effects of BDE-209 exposure on sexual development, genital gland development, sperm development and reproductive endocrine function in male rats before and after puberty.
METHODS: Twenty-four SD male rats (4 weeks old) were randomly divided into four groups. The rats were given different concentrations of BDE-209 (0,2,10,50 mg/kg.d) by gastric Administration for one month.
Result:
1. Compared with the control group, the activities of SOD, GSH-Px and AKP in testis of 50 mg/kg BDE-209 poisoning group were significantly lower than those of the control group (P 0.05). The pathological section of testis showed vacuole-like changes.
2. After BDE-209 exposure, the sperm motility decreased significantly with the increase of BDE-209 concentration (P 0.05), especially in the 50mg/kg group (P 0.05). Meanwhile, the sperm deformity rate of BDE-209 exposed group was significantly higher than that of the control group (P 0.05). However, there was no significant change in sperm density after exposure to BDE-209.
3. There were no significant changes in serum testosterone, serum estradiol and testosterone content in testicular tissue between BDE-209 and control group (P 0.05).
CONCLUSION: BDE-209 exposure before and after puberty has a certain effect on the morphology and function of rat testis, especially on the process of sperm differentiation and maturation, resulting in an increase in the rate of sperm deformity, a decrease in sperm motility and a decrease in sperm motility.
The third part is the study of the toxicity of Sertoli cells exposed to ten brominated diphenyl ethers in vitro.
Objective: To investigate the toxic effects of BDE-209 on Sertoli cells.
METHODS: Sertoli cells from 18-day-old SD rats were cultured for 24 hours after exposure to BDE-209. MTT assay was used to determine the concentration of Sertoli cells and Annexin V-FITC kit was used to detect the apoptosis of Sertoli cells.
Result:
1. Rat Sertoli cells entered the logarithmic growth phase after 36 hours of culture, and 48-72 hours was the appropriate exposure window period. After 24 hours of exposure to different concentrations of BDE-209, the cell survival rate decreased with the increase of the concentration of BDE-209. The relative survival rate of Sertoli cells decreased significantly when the concentration of BDE-209 was greater than 30 ug/ml (P 0.05).
2. Compared with the control group, the activity of T-AOC and SOD in Sertoli cells of BDE-209 exposed group showed a downward trend. The activity of T-AOC and SOD in 10,20 ug/ml exposed group was significantly lower than that in the control group (P 0.05).
3. After 24 hours of BDE-209 exposure, the apoptotic rate of Sertoli cells increased. When the concentration of BDE-209 was more than 20 ug/ml, the apoptotic rate of Sertoli cells increased significantly (P 0.05).
Conclusion: BDE-209 has cytotoxic effect on Sertoli cells cultured in vitro, and BDE-209 exposure can induce apoptosis of Sertoli cells.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R114

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