發(fā)布時(shí)間：2018-02-02 20:32

  本文關(guān)鍵詞： 鄰苯二甲酸二(2-乙基己基)酯 突觸發(fā)育 甲狀腺激素　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景鄰苯二甲酸酯(phthalic acid esters,PAEs)作為增塑劑被廣泛應(yīng)用于食品包裝袋、兒童玩具、醫(yī)療器材、化妝品等產(chǎn)品中,由于PAEs與其他基質(zhì)分子間為非共價(jià)結(jié)合,易于從產(chǎn)品中逸出,遷移至周邊環(huán)境中,人群暴露狀況嚴(yán)峻。PAEs具有抗雄激素和微弱的擬雌激素作用,可引起生殖與發(fā)育毒性,是公認(rèn)的環(huán)境內(nèi)分泌干擾物。近年發(fā)現(xiàn)PAEs對(duì)甲狀腺激素亦具有干擾作用。PAEs中以鄰苯二甲酸二(2-乙基己基)酯[di(2-ethylexyl)phthalate,DEHP]應(yīng)用最為廣泛,DEHP能夠通過(guò)胎盤(pán)屏障和血腦屏障,因此其神經(jīng)發(fā)育毒性備受關(guān)注。已有相關(guān)研究證實(shí),DEHP的性激素干擾特性可能在其所致神經(jīng)發(fā)育損傷中發(fā)揮作用。然而,從甲狀腺內(nèi)分泌干擾方面探討DEHP神經(jīng)毒性機(jī)制的研究卻鮮有報(bào)道。研究目的探討DEHP甲狀腺激素干擾特性在其所致突觸發(fā)育損害中的作用,為探討DEHP神經(jīng)毒性機(jī)制提供新思路。研究方法取自然分娩ICR小鼠,共14窩,每窩于出生后4天調(diào)整為5只雄鼠,同窩仔鼠隨機(jī)分為空白對(duì)照組、溶劑對(duì)照組以及DEHP處理組(10、50、200mg/kg·d),每組14只。空白對(duì)照組給予超純水,溶劑對(duì)照組給予含1%Tween80與1%玉米油的乳濁液,DEHP處理組給予按前述溶劑配置的DEHP乳濁液。出生后5天開(kāi)始按20μL/g體重對(duì)仔鼠進(jìn)行灌胃給藥,每日1次,至出生后38天。期間每天稱(chēng)量并記錄小鼠體重,出生后21天離乳后稱(chēng)量各組小鼠每日平均飼料消耗量及飲水量。出生后26天進(jìn)行曠場(chǎng)實(shí)驗(yàn),評(píng)估小鼠自發(fā)性探索活性及焦慮狀態(tài);出生后30天開(kāi)始進(jìn)行Morris水迷宮實(shí)驗(yàn),前5d進(jìn)行定位航行測(cè)試,間隔2d,第8d進(jìn)行空間探索測(cè)試,評(píng)估小鼠空間學(xué)習(xí)與記憶能力。行為學(xué)實(shí)驗(yàn)結(jié)束48h后,應(yīng)用10%水合氯醛溶液按0.04m L/10g體重進(jìn)行腹腔注射麻醉,采用摘眼球取血,分離血清,應(yīng)用直接化學(xué)發(fā)光法于全自動(dòng)生化分析儀上測(cè)定血清甲狀腺激素(T3、T4、FT3、FT4)及TSH水平;取雙側(cè)睪丸組織并稱(chēng)重;經(jīng)心臟行生理鹽水灌注后斷頭,取小腦組織,應(yīng)用LC-MS檢測(cè)DEHP主要一級(jí)代謝產(chǎn)物MEHP濃度;取單側(cè)大腦半球,應(yīng)用電化學(xué)發(fā)光法檢測(cè)腦組織T3與T4水平;分離單側(cè)大腦海馬組織,應(yīng)用蛋白免疫印跡法檢測(cè)突觸后致密蛋白95(postsynaptic density protein 95,PSD95)、突觸素I(synapsin I)、腦源性神經(jīng)營(yíng)養(yǎng)因子(brain-derived neurotrophic factor,BDNF)、甲狀腺激素受體α1(thyroid hormone receptorα1,TRα1)、甲狀腺激素受體β1(thyroid hormone receptorβ1,TRβ1)、單羧酸轉(zhuǎn)運(yùn)體8(monocarboxylate transporter 8,MCT8)、有機(jī)陰離子轉(zhuǎn)運(yùn)多肽1C1(organic anion transporting polypeptide 1C1,OATP1C1)、II型脫碘酶(deiodinase Ⅱ,DIO2)、Ⅲ型脫碘酶(deiodinase Ⅲ,DIO3)蛋白水平。結(jié)果各項(xiàng)指標(biāo)在溶劑對(duì)照組與空白對(duì)照組之間均無(wú)明顯差異(皆P0.05)。200mg/kg·d DEHP組小鼠體重增加減少、睪丸臟器系數(shù)下降、小腦組織中MEHP水平升高(與溶劑對(duì)照組比較,皆P0.05),表明DEHP可能引起了明顯的抗雄激素效應(yīng),且能通過(guò)血腦屏障。各組小鼠在曠場(chǎng)實(shí)驗(yàn)和水迷宮實(shí)驗(yàn)空間探索階段中總運(yùn)動(dòng)距離均無(wú)明顯差異(皆P0.05),表明本次研究劑量范圍DEHP未明顯影響小鼠運(yùn)動(dòng)能力。曠場(chǎng)實(shí)驗(yàn)中,200mg/kg·d DEHP組小鼠曠場(chǎng)中央?yún)^(qū)運(yùn)動(dòng)時(shí)間減少(與溶劑對(duì)照組比較,P0.05),而10與50mg/kg·d DEHP組小鼠與溶劑對(duì)照組比較無(wú)明顯差異(P0.05),表明10與50mg/kg·d DEHP未明顯引起小鼠焦慮情緒。Morris水迷宮實(shí)驗(yàn)中,200mg/kg·d DEHP組小鼠登臺(tái)潛伏期延長(zhǎng)(與溶劑對(duì)照組比較,P0.05);空間探索階段,50mg/kg·d DEHP組小鼠目標(biāo)象限運(yùn)動(dòng)距離及時(shí)間減少(與溶劑對(duì)照組比較,P0.05),表明50mg/kg·d DEHP能夠損傷小鼠空間記憶能力。DEHP各劑量組小鼠海馬PSD95蛋白水平均降低(與溶劑對(duì)照組比較,皆P0.05),200mg/kg·d DEHP組還觀察到小鼠海馬synapsin I蛋白水平降低(與溶劑對(duì)照組比較,P0.05),表明DEHP可損害小鼠突觸發(fā)育。各組血清T3、T4、FT3、FT4、TSH水平及腦組織T3、T4水平均無(wú)明顯差異(皆P0.05),而200mg/kg·d DEHP組小鼠腦組織T4/T3比值明顯升高(與溶劑對(duì)照組比較,P0.05),表明DEHP雖然未影響循環(huán)中甲狀腺素穩(wěn)態(tài),但高劑量可引起大腦T4和T3的相對(duì)平衡。DEHP各劑量組小鼠海馬OATP1C1蛋白水平均降低(與溶劑對(duì)照組比較,皆P0.05),10mg/kg·d DEHP組還觀察到海馬MCT8蛋白水平降低(與溶劑對(duì)照組比較,P0.05),各組小鼠海馬DIO2與DIO3蛋白水平均無(wú)明顯差異(皆P0.05),提示DEHP可能會(huì)影響海馬甲狀腺激素轉(zhuǎn)運(yùn)。海馬甲狀腺素受體的檢測(cè)發(fā)現(xiàn),200mg/kg·d DEHP組的TRα1和TRβ1蛋白水平均降低(與溶劑對(duì)照組比較,皆P0.05),需要特別注意的是,50mg/kg·d DEHP組TRβ1蛋白水平就開(kāi)始降低(與溶劑對(duì)照組比較,P0.05),提示在受體水平,DEHP可能首先影響TRβ1。200mg/kg·d DEHP組小鼠海馬BDNF蛋白水平降低(與溶劑對(duì)照組比較,P0.05)。結(jié)論⑴發(fā)育期暴露較高劑量DEHP(200mg/kg·d)能夠影響小鼠一般發(fā)育,可能引起了明顯的抗雄激素效應(yīng),能夠損害海馬突觸發(fā)育,引起焦慮情緒并降低空間學(xué)習(xí)能力,能夠引起大腦甲狀腺激素內(nèi)穩(wěn)態(tài)失衡,并可能干擾了甲狀腺激素受體信號(hào);⑵發(fā)育期暴露較低劑量DEHP(50mg/kg·d)雖然未明顯影響小鼠一般發(fā)育、未引起明顯的焦慮情緒,但能夠損害小鼠海馬突觸發(fā)育,降低空間記憶能力,可能干擾了海馬局部甲狀腺激素內(nèi)穩(wěn)態(tài)及其受體信號(hào);綜上所述,發(fā)育期暴露DEHP,在不明顯影響小鼠一般發(fā)育、不引起焦慮情緒的劑量條件下,即可損害小鼠海馬突觸發(fā)育,降低空間記憶能力。DEHP影響甲狀腺激素轉(zhuǎn)運(yùn)及其受體水平可能干擾了海馬局部甲狀腺激素內(nèi)穩(wěn)態(tài)及其受體信號(hào),進(jìn)而損害海馬突觸發(fā)育。
[Abstract]:On the background of the adjacent benzene two carbamate (phthalic acid esters, PAEs) as the plasticizer is widely used in food packaging bags, children's toys, medical equipment, cosmetics and other products, because of the PAEs and other matrix molecules for non covalent binding, easy to escape from the product, migrated to the surrounding environment, population exposure status severe.PAEs with anti androgen and weak estrogenic effect, can cause reproductive and developmental toxicity, environmental endocrine disruptors is recognized. In recent years PAEs also has the interference effects of.PAEs in the adjacent benzene two formic acid two of thyroid hormone (2- ethylhexyl) ester [di (2-ethylexyl) phthalate, DEHP] is the most widely used, DEHP through the placental barrier and the blood brain barrier, so the neural developmental toxicity has attracted much attention. Studies have confirmed that the sex hormone disturbance characteristics of DEHP may play a role in the development of the nerve injury caused by it. However, study the mechanism of neurotoxicity of DEHP from thyroid endocrine disruption is rarely reported. Objective to study the DEHP characteristics of thyroid hormone disrupting role in synaptic development caused by damage in, to provide new ideas for explore the mechanism of neurotoxicity of DEHP. Research methods of natural childbirth ICR mice, a total of 14 nests per litter was born 4 days after adjustment 5 male rats, littermate rats were randomly divided into blank control group, solvent control group and DEHP treatment group (10,50200mg/kg, d), 14 rats in each group. The control group was given ultrapure water, solvent control group was treated with 1%Tween80 and 1% corn oil emulsion, DEHP treatment group was given according to the configuration of the DEHP solvent emulsion. 5 days after birth to 20 L/g body weight of rats were administered orally, 1 times a day to 38 days after birth. During the day and record the weight weighing mice 21 days after birth, weaning after weighing the mice per group The average daily feed and water consumption. The open field experiment was carried out 26 days after birth, spontaneous activity and exploration evaluation anxiety; born 30 days after Morris water maze test, 5D positioning navigation test, spatial probe test interval 2D, 8D, spatial learning and memory ability of mice to evaluate the behavior. The 48h after the application of 10% chloral hydrate with 0.04m weight of L/10g were anesthetized with intraperitoneal injection, the eyeball blood, separation of serum, the automatic biochemical analyzer for determination of serum thyroid hormone application of direct chemiluminescence method (T3, T4, FT3, FT4) and the level of TSH; bilateral testicular tissue and weighed. The heart; saline infusion after decapitation, the cerebellum, the application of LC-MS for detection of DEHP main product level metabolism MEHP concentration; take unilateral cerebral hemisphere and the application of chemiluminescence detection of brain tissue T3 and T4 levels; separation The brain tissue of unilateral hippocampal synaptic detection, using the Western blot method after protein 95 (postsynaptic density compact protein 95, PSD95), I (synapsin I), synaptophysin brain-derived neurotrophic factor (brain-derived neurotrophic, factor, BDNF), thyroid hormone receptor alpha 1 (thyroid hormone receptor TR alpha 1, alpha 1). Thyroid hormone receptor 1 (thyroid hormone receptor TR beta 1, beta 1), monocarboxylate transporter (monocarboxylate 8 transporter 8, MCT8), organic anion transport polypeptide 1C1 (organic anion transporting polypeptide 1C1, OATP1C1), type II deiodinase (deiodinase II, DIO2), type III deiodinase (deiodinase III DIO3), protein level. The results of the indicators in the solvent control group and blank control group had no significant difference (all P0.05),.200mg/kg D DEHP group were added to reduce body weight, decrease the organ coefficient of testis, elevated levels of MEHP in cerebellum (and The solvent control group, all P0.05), indicating that DEHP may cause the anti androgen effect is obvious, and can pass through the blood brain barrier. Mice in the open field test and water maze test showed no significant difference between the total space exploration stage movement (all P0.05), in the distance that the study dose range of DEHP did not significantly influence exercise capacity of mice. The open field test, 200mg/kg and D in DEHP group reduced exercise time (central open field compared to the control group, and P0.05, and the 10 solvent) and 50mg/kg D in DEHP group and solvent control group had no significant difference (P0.05), D DEHP and 50mg/kg 10 showed no obvious cause of anxiety in mice the mood in.Morris water maze, 200mg/kg and D in DEHP group on latency (control group, solvent and P0.05); space exploration stage, 50mg/kg and D in DEHP group exercise time and reduce the distance of the target quadrant (control group, P0. and solvent 05), showed that 50mg/kg D DEHP can damage the spatial memory ability of mice.DEHP mice hippocampal PSD95 protein levels were decreased (control group, P0.05, 200mg/kg and solvent are) d group DEHP was also observed in the hippocampal synapsin protein level of I decreased (compared to the control group, and P0.05, DEHP can show that solvent) the damage of mouse synaptic development. The serum concentrations of T3, T4, FT3, FT4, TSH and T3 in brain tissue, there were no obvious differences in the level of T4 (all P0.05), and 200mg/kg D DEHP mice brain tissue T4/T3 ratio increased significantly (compared to the control group, and P0.05, DEHP showed that although the solvent) did not affect circulating thyroxine the steady state, but high dose can cause the relative balance of.DEHP brain T4 and T3 mice hippocampus OATP1C1 protein levels were decreased (control group, P0.05, 10mg/kg and solvent are) d group DEHP was also observed in hippocampal MCT8 protein level decreased (with solvent than the control group A, P0.05), there were no significant difference between the groups of mice hippocampus DIO2 and DIO3 protein level (all P0.05), suggesting that DEHP may affect the hippocampus. Detection of thyroid hormone transport hormone receptor in the hippocampus, 200mg/kg and D in DEHP group and TR TR alpha 1 beta 1 protein levels decreased (with the solvent control group, is P0.05), special attention is needed, the 50mg/kg D DEHP group TR beta 1 protein level began to decrease (compared to the control group, and the solvent P0.05), suggesting that at the receptor level, DEHP may be the first effect of TR beta 1.200mg/kg D DEHP mice BDNF protein level in the hippocampus decreased (compared to the control group and solvent P0.05). Conclusions the development period of exposure to higher doses of DEHP (200mg/kg - D) can affect the MICE development, may cause the anti androgen effect is obvious, can damage hippocampal synaptic growth caused anxiety and spatial learning ability is reduced, can cause brain thyroid hormone In the homeostasis, and may interfere with thyroid hormone receptor signal; the development period with lower exposure dose of DEHP (50mg/kg, d) although not significantly affect the general development of mice, did not cause obvious anxiety, but can damage hippocampal synaptic growth, reduce the spatial memory ability, may interfere with thyroid hormone local steady-state in hippocampus. To sum up, and its receptor signal; developmental exposure to DEHP, no obvious effect in mice development, does not cause dose anxiety, can damage hippocampal synaptic growth, reduce the spatial memory ability of.DEHP transport and the effects of thyroid hormone receptor levels may interfere with thyroid hormone local homeostasis in the hippocampus and its receptor signaling, and hippocampal synaptic damage development.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R114

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