发育期DEHP暴露通过干扰大脑甲状腺激素诱导小鼠海马突触发育损害的实验研究

发布时间：2018-02-02 20:32

本文关键词： 邻苯二甲酸二(2-乙基己基)酯突触发育甲状腺激素　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景邻苯二甲酸酯(phthalic acid esters,PAEs)作为增塑剂被广泛应用于食品包装袋、儿童玩具、医疗器材、化妆品等产品中,由于PAEs与其他基质分子间为非共价结合,易于从产品中逸出,迁移至周边环境中,人群暴露状况严峻。PAEs具有抗雄激素和微弱的拟雌激素作用,可引起生殖与发育毒性,是公认的环境内分泌干扰物。近年发现PAEs对甲状腺激素亦具有干扰作用。PAEs中以邻苯二甲酸二(2-乙基己基)酯[di(2-ethylexyl)phthalate,DEHP]应用最为广泛,DEHP能够通过胎盘屏障和血脑屏障,因此其神经发育毒性备受关注。已有相关研究证实,DEHP的性激素干扰特性可能在其所致神经发育损伤中发挥作用。然而,从甲状腺内分泌干扰方面探讨DEHP神经毒性机制的研究却鲜有报道。研究目的探讨DEHP甲状腺激素干扰特性在其所致突触发育损害中的作用,为探讨DEHP神经毒性机制提供新思路。研究方法取自然分娩ICR小鼠,共14窝,每窝于出生后4天调整为5只雄鼠,同窝仔鼠随机分为空白对照组、溶剂对照组以及DEHP处理组(10、50、200mg/kg·d),每组14只。空白对照组给予超纯水,溶剂对照组给予含1%Tween80与1%玉米油的乳浊液,DEHP处理组给予按前述溶剂配置的DEHP乳浊液。出生后5天开始按20μL/g体重对仔鼠进行灌胃给药,每日1次,至出生后38天。期间每天称量并记录小鼠体重,出生后21天离乳后称量各组小鼠每日平均饲料消耗量及饮水量。出生后26天进行旷场实验,评估小鼠自发性探索活性及焦虑状态;出生后30天开始进行Morris水迷宫实验,前5d进行定位航行测试,间隔2d,第8d进行空间探索测试,评估小鼠空间学习与记忆能力。行为学实验结束48h后,应用10%水合氯醛溶液按0.04m L/10g体重进行腹腔注射麻醉,采用摘眼球取血,分离血清,应用直接化学发光法于全自动生化分析仪上测定血清甲状腺激素(T3、T4、FT3、FT4)及TSH水平;取双侧睾丸组织并称重;经心脏行生理盐水灌注后断头,取小脑组织,应用LC-MS检测DEHP主要一级代谢产物MEHP浓度;取单侧大脑半球,应用电化学发光法检测脑组织T3与T4水平;分离单侧大脑海马组织,应用蛋白免疫印迹法检测突触后致密蛋白95(postsynaptic density protein 95,PSD95)、突触素I(synapsin I)、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、甲状腺激素受体α1(thyroid hormone receptorα1,TRα1)、甲状腺激素受体β1(thyroid hormone receptorβ1,TRβ1)、单羧酸转运体8(monocarboxylate transporter 8,MCT8)、有机阴离子转运多肽1C1(organic anion transporting polypeptide 1C1,OATP1C1)、II型脱碘酶(deiodinase Ⅱ,DIO2)、Ⅲ型脱碘酶(deiodinase Ⅲ,DIO3)蛋白水平。结果各项指标在溶剂对照组与空白对照组之间均无明显差异(皆P0.05)。200mg/kg·d DEHP组小鼠体重增加减少、睾丸脏器系数下降、小脑组织中MEHP水平升高(与溶剂对照组比较,皆P0.05),表明DEHP可能引起了明显的抗雄激素效应,且能通过血脑屏障。各组小鼠在旷场实验和水迷宫实验空间探索阶段中总运动距离均无明显差异(皆P0.05),表明本次研究剂量范围DEHP未明显影响小鼠运动能力。旷场实验中,200mg/kg·d DEHP组小鼠旷场中央区运动时间减少(与溶剂对照组比较,P0.05),而10与50mg/kg·d DEHP组小鼠与溶剂对照组比较无明显差异(P0.05),表明10与50mg/kg·d DEHP未明显引起小鼠焦虑情绪。Morris水迷宫实验中,200mg/kg·d DEHP组小鼠登台潜伏期延长(与溶剂对照组比较,P0.05);空间探索阶段,50mg/kg·d DEHP组小鼠目标象限运动距离及时间减少(与溶剂对照组比较,P0.05),表明50mg/kg·d DEHP能够损伤小鼠空间记忆能力。DEHP各剂量组小鼠海马PSD95蛋白水平均降低(与溶剂对照组比较,皆P0.05),200mg/kg·d DEHP组还观察到小鼠海马synapsin I蛋白水平降低(与溶剂对照组比较,P0.05),表明DEHP可损害小鼠突触发育。各组血清T3、T4、FT3、FT4、TSH水平及脑组织T3、T4水平均无明显差异(皆P0.05),而200mg/kg·d DEHP组小鼠脑组织T4/T3比值明显升高(与溶剂对照组比较,P0.05),表明DEHP虽然未影响循环中甲状腺素稳态,但高剂量可引起大脑T4和T3的相对平衡。DEHP各剂量组小鼠海马OATP1C1蛋白水平均降低(与溶剂对照组比较,皆P0.05),10mg/kg·d DEHP组还观察到海马MCT8蛋白水平降低(与溶剂对照组比较,P0.05),各组小鼠海马DIO2与DIO3蛋白水平均无明显差异(皆P0.05),提示DEHP可能会影响海马甲状腺激素转运。海马甲状腺素受体的检测发现,200mg/kg·d DEHP组的TRα1和TRβ1蛋白水平均降低(与溶剂对照组比较,皆P0.05),需要特别注意的是,50mg/kg·d DEHP组TRβ1蛋白水平就开始降低(与溶剂对照组比较,P0.05),提示在受体水平,DEHP可能首先影响TRβ1。200mg/kg·d DEHP组小鼠海马BDNF蛋白水平降低(与溶剂对照组比较,P0.05)。结论⑴发育期暴露较高剂量DEHP(200mg/kg·d)能够影响小鼠一般发育,可能引起了明显的抗雄激素效应,能够损害海马突触发育,引起焦虑情绪并降低空间学习能力,能够引起大脑甲状腺激素内稳态失衡,并可能干扰了甲状腺激素受体信号;⑵发育期暴露较低剂量DEHP(50mg/kg·d)虽然未明显影响小鼠一般发育、未引起明显的焦虑情绪,但能够损害小鼠海马突触发育,降低空间记忆能力,可能干扰了海马局部甲状腺激素内稳态及其受体信号;综上所述,发育期暴露DEHP,在不明显影响小鼠一般发育、不引起焦虑情绪的剂量条件下,即可损害小鼠海马突触发育,降低空间记忆能力。DEHP影响甲状腺激素转运及其受体水平可能干扰了海马局部甲状腺激素内稳态及其受体信号,进而损害海马突触发育。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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