环境雌激素壬基酚对仔鼠神经发育毒性及机制研究

发布时间：2018-05-01 05:00

本文选题：壬基酚 + 仔鼠　；参考：《重庆医科大学》2012年博士论文

【摘要】：壬基酚(Nonylphenol, NP)是环境雌激素样化学物的典型代表，，作为乳化剂广泛应用于工业、农业、日用品生产中，可由污水排入水体环境。NP在环境中难于降解，具有高度脂溶性和生物蓄积性，可经口腔、呼吸道、皮肤等多种途径进入机体，对人群健康产生危害。研究估计，非职业暴露人群通过各种途径接受NP的暴露水平为3.6～31.4μg/kg/day。体内、外实验显示NP的雌激素活性约为雌二醇的10-4～10-5倍，可模拟雌激素与机体内多组织器官的雌激素受体结合发挥雌激素样作用，目前较多关于NP生殖和发育毒性的研究表明，NP的毒性机制主要源于其对内分泌的干扰作用，可对鱼类、两栖动物及哺乳动物多种器官产生不良作用：包括影响生殖、内分泌、免疫、消化、泌尿等系统的功能，然而NP对子代神经行为发育的影响目前尚不清楚。中枢神经系统在发育时期对体内多种激索水平的变化非常敏感，相对成人而言，胎儿在胚胎中后期和哺乳期，血脑屏障未发育成熟，中枢神经系统正值器官形成期，此时期最易受外来毒物损害，其神经系统器官发育和功能很可能受到影响。因此，若机体此期间若暴露于内分泌干扰物NP，可能导致胎儿神经系统所受的损害会比其它时期更为严重。以往研究大多是多种环境内分泌干扰物(EnvironmentalEndocrine Disrupters,EEDs)的联合暴露，不能说明NP单独的毒性作用，得出的结果也各不相同，因此结论尚具有不确定性，对机理的研究较片面，缺乏深入而系统的研究。本课题以大鼠为研究对象，首次在神经系统胚胎发育敏感期暴露NP，观察NP对仔鼠神经行为发育的影响，测试其学习记忆能力的变化，并从细胞凋亡、神经营养因子、胶质细胞纤维酸性蛋白、信号传递等角度进行研究，同时利用基因芯片从分子水平尝试探讨NP神经毒性的分子机制，进而可为NP在发育期间暴露的危害评价提供实验数据，为制定相关环境污染标准提供理论依据。第一部分NP对母鼠生殖和仔鼠生长发育的影响目的:研究NP对雌性大鼠生产和仔鼠生长发育的影响。方法：SD孕鼠28只随机分为4组（对照组、50mg/kg NP、100mg/kgNP、200mg/kg NP），每组6～7只，妊娠第9～15天灌胃NP，观察孕鼠的体重、进食、染毒后的一般情况以及生殖情况，观察仔鼠的出生指标及体重变化，检测仔鼠早期生理发育指标。结果：（1）各实验组孕鼠末见明显中毒症状，100mg/kg（中剂量组）和200mg/kg（高剂量组）NP组孕鼠每窝平均胎数、活胎数降低，死胎数增加，p0.05。（2）高剂量组仔鼠身长、尾长、肛殖距缩短，p0.05。（3）各实验组仔鼠出生脏器重量（脑，肝、肾、心等）与对照组比较差异无统计学意义，p0.05。（4）高剂量组体重出生后1、7、14、21、28天均低于对照组，p0.01。（5）高剂量组仔鼠早期生理发育指标（张耳、开眼、长毛、出牙）发育时间较对照组延长，p0.05。以上指标低剂量组与对照组比较差异均无统计学意义，p0.05。结论:在本实验的染毒方式、时间和剂量下，高剂量组NP对雌鼠的生殖能力有影响，同时可影响仔鼠的出生指标和生长发育指标。第二部分NP对仔鼠神经行为发育的影响目的:探讨母鼠NP孕期暴露致子代神经行为发育及学习记忆的影响。方法:孕鼠染毒及分组方法同第一部分。在出生后特定的时间，观察仔鼠断崖回避、平面翻正、前肢悬挂、空中翻正、听觉惊愕和视觉定位等早期神经行为发育指标,选择8周龄仔鼠进行Morris水迷宫和跳台试验，检测NP对其学习记忆的影响；脱椎取脑组织做石蜡切片HE染色光镜下观察其病理变化；海马组织透射电镜观察超微结构变化。结果:（1）早期神经行为发育指标：高剂量组仔鼠（断崖回避、平面翻正、前肢悬挂、空中翻正、听觉惊愕和视觉定位等）时间较对照组延长（p0.05）；（2）Morris水迷宫试验：高剂量组仔鼠水迷宫中逃避潜伏期延长(p005)；（3）跳台试验：反应时间延长，步下潜伏期缩短，错误次数增加(p005)；（4）病理：HE染色，光镜可见高暴露组海马组织充血水肿；（5）透射电镜：高剂量组仔鼠海马组织电镜下见线粒体肿胀呈空泡样变，染色质浓缩成块状聚集在核周围。结论: NP孕期暴露可阻碍仔鼠早期神经行为的发育，使空间学习记忆能力下降。第三部分NP对断乳期仔鼠海马基因表达谱的影响目的：应用基因芯片技术，考察NP暴露组和对照组仔鼠海马组织差异基因的表达状况，筛选部分与神经毒性相关的差异表达基因及通路进行进一步机制研究。方法：建立孕期和哺乳期暴露NP的仔鼠模型，提取暴露组和对照组出生1天仔鼠海马组织mRNA，采用Roche Nimblegen公司生产的12×135位点的包含大鼠26,419个基因的表达谱基因芯片，检测脑基因的表达，扫描仪进行扫描及处理数据。结果：暴露组与对照组共有1,254个差异表达基因，其中619个基因上调，635个基因下调。部分与神经系统功能相关差异表达基因为：A.神经营养相关的基因表达：甘丙肽(Galanin，Gal)基因下调，神经生长因子(Nerve Growth Factor，NGF)基因下调；B.细胞凋亡的相关基因的表达：凋亡蛋白酶活化因子-1(Apoptotic Protease Activating Factor-1,Apaf-1)基因上调，凋亡抑制基因-1（Defender Against Apoptotic Death-1，DAD1）基因下调，半胱氨酸蛋白酶7(Cysteine-aspartic Acid Protease，Caspase7)基因上调；C.信号传递及离子通道相关基因的表达：谷氨酸盐受体(Glutamate Receptor, Ionotropic,AMPA2)基因下调，钙调素依赖性蛋白激酶Ⅱ (Calcium/calmodulin-dependent Protein Kinase II Delta,Camk2d)基因下调。结论:芯片结果提示： NP能导致仔鼠海马神经元信号传导、免疫应答、细胞凋亡、炎症反应因子、神经胶质细胞发育等相关基因的差异表达，以上改变有可能干扰神经系统的发育和功能的发挥。第四部分NP暴露对仔鼠神经发育毒性的机制研究目的：母鼠孕期及哺乳期暴露NP，观察对子代神经细胞凋亡、神经递质、胶质细胞以及神经生长相关基因的表达变化，探讨NP对仔鼠神经毒性的可能机制。方法：将交配成功的31只孕鼠根据妊娠日期分层后随机分配到4组，即C、L、M、H组（NP0、25、50、100mg/kg/day），单笼饲养，空腹灌胃, NP暴露时间为受孕第6天到出生后21天哺乳期结束（GD6～PND21），放免法检测21天仔鼠血清雌二醇(E2)和睾酮(TT)水平；分光比色法测仔鼠海马组织胆碱乙酰转移酶（ChAT）和胆碱酯酶（AchE）活性；免疫组化发观察海马组织即早基因c-fos和c-jun蛋白的表达；原位末端标记法(TUNEL)观察PND21天和60天仔鼠海马神经元凋亡发生情况；免疫组织化学法检测海马和皮质星形胶质细胞胶质原纤维酸性蛋白(GFAP)、神经生长相关因子(GAP-43)的蛋白的变化，RealTime PCR法检测GFAP和GAP-43mRNA的变化，并分析上述指标变化与NP暴露剂量之间的关系。结果：①NP对断乳期仔鼠血清激素水平的影响：放免法检测结果显示，仔鼠出生后21天血清睾酮水平随NP暴露剂量增加而降低，存在剂量-效应关系（r=-0.889，p 0.05），与对照组比较，M和H两个剂量组睾酮明显降低（p0.05）；雌激素水平与NP暴露剂量正相关（r=-0.462，p0.01），H剂量组雌激素水平与对照组比较差异有统计学意义（p 0.05）。②NP暴露对断乳期及成熟期仔鼠神经细胞凋亡的影响：TUNEL法检测结果显示，仔鼠出生后21d和60d，H剂量组海马细胞凋亡率显著高于对照组(p 0.01)，且NP暴露剂量与凋亡率正相关（r=0.836、0.521，p 0.05）。从纵向比较，各暴露组21天仔鼠海马组织凋亡率均高于60天（t=3.331，p0.05）③NP对仔鼠神经信号传导的影响：A. NP对仔鼠海马组织ChAT和AchE活性的影响：M、H剂量组仔鼠海马组织ChAT活性明显低于对照组；M剂量组AchE活性高于对照组（p 0.01）。海马组织ChAT和AchE活性与NP暴露剂量有剂量效应关系（r=-0.821、0.757，p 0.05）。B.海马组织c-fos和c-jun蛋白表达的变化：正常仔鼠海马中c-jun和c-fos蛋白表达很低，L剂量组阳性细胞数少，着色浅；M和H剂量组仔鼠海马c-jun和c-fos阳性细胞数量增多，尤其是高剂量组分布密集，胞体大、着色深。④NP对仔鼠海马星形胶质细胞胶质原纤维酸性蛋白的影响：免疫组化技术检测GFAP蛋白表达的结果显示，H剂量组21d和60d龄的仔鼠海马和皮质部GFAP免疫阳性反应细胞数目、积分光密度显著高于同期对照组仔鼠(p 0.05)，荧光定量PCR检测GFAP表达的结果显示，H剂量组21d和60d龄的仔鼠海马GFAP mRNA表达升高，与对照组比较有显著性差异。GFAP蛋白与GFAP mRNA表达与NP暴露剂量有正相关关系(p 0.05)。⑤NP对仔鼠神经生长相关蛋白的影响：免疫组化技术检测GAP-43蛋白表达的结果显示，H剂量组21d和60d龄的仔鼠海马和皮质部GAP-43免疫反应阳性细胞平均数目、积分光密度低于同期对照组仔鼠(p 0.05)，荧光定量PCR结果显示，H剂量组21d和60d龄的仔鼠海马GAP-43mRNA表达下降，差异有统计学意义。GAP-43蛋白和GAP-43mRNA表达与NP暴露剂量有负相关关系(p 0.05)。结论：结合以上数据，推测NP诱导的神经毒性可能机制是：在胚胎期及哺乳期接触NP， NP的弱雌激素样作用通过竞争雌激素受体引起内分泌系统失衡，导致仔鼠体内雌激素水平升高，改变了子代大脑发育的内分泌环境，干扰神经细胞发育过程，降低神经营养因子GAP-43的表达水平，抑制神经细胞分化，包括神经突起生长和分支，突触的形成，同时通过增加胶质细胞纤维酸性蛋白GFAP表达，并影响星形胶质细胞的形态、结构和功能，进一步影响胆碱能神经递质Ach以及即早基因c-jun、c-fos的信息传递，并且诱导仔鼠海马区神经细胞凋亡，最终导致子代发育期神经反射时间延迟，成熟期学习记忆障碍。
[Abstract]:Nonylphenol (NP) is a typical representative of environmental estrogenic chemicals. As a emulsifier, it is widely used in industrial, agricultural and daily necessities production. It can be discharged into the water environment by sewage and.NP is difficult to degrade in the environment. It has high fat solubility and bioaccumulation, and can enter the body through oral, respiratory, skin and other ways. Health is harmful.
It is estimated that the exposure level of non occupational exposures to NP is 3.6 to 31.4 micron g/kg/day. in various ways. The external experiment shows that the estrogen activity of NP is about 10-4 to 10-5 times as much as estradiol. Developmental toxicity studies have shown that the toxic mechanism of NP mainly originates from its endocrine disruption and has adverse effects on various organs of fish, amphibians and mammals, including the functions of reproductive, endocrine, immune, digestive, and urinary systems. However, the effects of NP on neurobehavioral development of the progeny are not yet clear.
The central nervous system is very sensitive to a variety of changes in the level of irritable cord in the body during the development period. Compared with the adult, the fetus is not mature in the middle and late embryo and lactation period, and the central nervous system is in the stage of organogenesis. This period is most vulnerable to foreign poison, and the development and function of the nervous system organs are likely to be affected. Therefore, if the body is exposed to the endocrine disruptor NP during this period, the damage to the fetal nervous system may be more serious than that of other periods. Previous studies were mostly combined exposure to a variety of environmental endocrine disruptors (EnvironmentalEndocrine Disrupters, EEDs), which could not explain the toxic effects of NP alone, and the results were also found. Therefore, the conclusion is still uncertain. The research on mechanism is rather one-sided and lacks in-depth and systematic research.
This subject takes rats as the research object, exposes NP in the sensitive stage of the embryonic development of the nervous system for the first time, observing the effect of NP on the neurobehavioral development of the offspring, testing the changes of learning and memory ability, and studying from the angle of cell apoptosis, neurotrophic factor, glial fibrillary acidic protein, signal signal transmission and so on, and using gene chip from the angle of gene chip. At the molecular level, we try to explore the molecular mechanism of NP neurotoxicity, and then provide experimental data for the risk assessment of NP exposure during development, and provide a theoretical basis for the formulation of related environmental pollution standards.
Part one the effect of NP on the growth and development of female rats and their offspring.
Objective: To study the effects of NP on the growth and development of female rats and offspring rats.
Methods: 28 SD pregnant rats were randomly divided into 4 groups (control group, 50mg/kg NP, 100mg/kgNP, 200mg/kg NP), 6~7 rats in each group, NP in each group for ninth to 15 days. The weight of pregnant rats, eating, general condition and reproductive status were observed. The birth index and weight change of the offspring were observed and the early physiological development indexes of the offspring were detected.
Results: (1) there were obvious poisoning symptoms at the end of the pregnant rats in the experimental groups. The average number of fetus per litter in 100mg/kg (medium dose group) and 200mg/kg (high dose group) NP group, the number of live births and the number of stillbirths increased, the length of the rats in the high dose group p0.05. (2), the tail length, the anal colonization shortened, and p0.05. (3) the weight of the viscera (brain, liver, kidney, heart, etc.) of the offspring rats of the experimental groups (3) and the control group. The difference was not statistically significant. The p0.05. (4) high dose group was lower than the control group at 1,7,14,21,28 days after birth, and the development time of early physiological development index (Zhang Er, eye opening, long hair, tooth) in the high dose group of p0.01. (5) high dose group was longer than that of the control group, and there was no statistical difference between the low dose group above the p0.05. index and the control group, p0.05.
Conclusion: at the time and dose of the experiment, the high dose group NP has an influence on the reproductive ability of the female rats, and it can also affect the birth index and the growth and development index of the offspring.
The effect of second part NP on the neurobehavioral development of offspring rats
Objective: To investigate the effects of NP pregnancy exposure on offspring's neurobehavioral development and learning and memory.
Methods: pregnant rats were infected and grouped in the same way as the first part. At the specific time after birth, the early neurobehavioral development indicators such as cliffs avoidance, plane reversal, forelimb suspension, air reversal, auditory consternation and visual orientation were observed and the effects of NP on the learning and memory of the 8 week old rats were tested by the Morris water fan palace and the platform test. The brain tissue was removed by paraffin section and stained with HE. The pathological changes were observed under light microscope. Ultrastructural changes were observed by transmission electron microscope.
Results: (1) early neurobehavioral development index: the time of high dose group (Cliffs avoidance, plane reversal, forelimb suspension, air reversal, auditory consternation and visual orientation) was longer than that of the control group (P0.05); (2) Morris water maze test: the escape latency prolonged (P005) in the water maze of high dose group rats (P005); (3) the jump test: reaction time delay Long, lower latency and increased number of errors (P005); (4) pathology: HE staining, light microscopy showed hyperemia in the hippocampus of high exposure group; (5) transmission electron microscopy: high dose group of hippocampus tissue under the electron microscope showed vacuolar like changes in mitochondria, chromatin concentrated around the nucleus.
Conclusion: exposure to NP during pregnancy can hinder the development of neural behavior and reduce the ability of spatial learning and memory.
The third part is the effect of NP on the gene expression profile in the hippocampus of weaning rats.
Objective: To investigate the expression of differentially expressed genes in the hippocampus of NP exposed group and control group by gene chip technology, and to select the differentially expressed genes and pathways related to neurotoxicity. Methods: to establish the offspring model of NP in pregnancy and lactation period, and to extract the 1 day offspring from the exposure group and the control group. The rat hippocampal tissue mRNA, using the 12 x 135 locus produced by Roche Nimblegen, contained the gene chip of 26419 genes of the rat, detected the expression of the brain gene, and scanned and processed the data by the scanner.
Results: there were 1254 differentially expressed genes in the exposure group and the control group, of which 619 genes were up-regulated and 635 genes were down. Some genes related to the function of the nervous system were expressed as: A. neurotrophic related gene expression: Galanin (Gal) gene downregulation, neural growth factor (Nerve Growth Factor, NGF) gene downregulation; B. The expression of apoptosis related genes: apoptotic protease activating factor -1 (Apoptotic Protease Activating Factor-1, Apaf-1) up-regulated, apoptosis inhibitory gene -1 (Defender Against Apoptotic Death-1, DAD1) gene regulation, cysteine protease 7 gene regulation; The expression of the ion channel related genes: the Glutamate Receptor (Ionotropic, AMPA2) gene was down regulated, and the calmodulin dependent protein kinase II (Calcium/calmodulin-dependent Protein Kinase II Delta, Camk2d) gene was downregulated.
Conclusion: the results of the chip suggest that NP can lead to differentially expressed genes such as signal transduction, immune response, apoptosis, inflammatory response factor, and glial cell development in the hippocampal neurons of the offspring, and the above changes may interfere with the development and function of the nervous system.
The fourth part is the mechanism of NP exposure on the neurodevelopmental toxicity of offspring rats.
Objective: To observe the changes of apoptosis, neurotransmitters, glial cells and nerve growth related genes in the pregnant and lactation period of NP rats, and to explore the possible mechanism of NP on the neurotoxicity of the offspring.
Methods: 31 pregnant mice were randomly assigned to 4 groups according to the date of pregnancy, namely, C, L, M, H group (NP0,25,50100mg/kg/day), single cage, fasting stomach, NP exposure time for sixth days of pregnancy to 21 days after birth (GD6 to PND21), and radioimmunoassay for the detection of serum estradiol (E2) and testosterone (TT) levels in the serum for 21 days. The activity of cholinesterase (ChAT) and cholinesterase (AchE) in hippocampal tissue of offspring was measured by colorimetric method. The expression of early gene c-fos and c-jun protein in hippocampus was observed by immunohistochemical staining. In situ terminal labeling method (TUNEL) was used to observe the apoptosis of hippocampal neurons in PND21 days and 60 days, and immunohistochemistry was used to detect hippocampus and cortex. The changes in the protein of glial glial fibrillary acidic protein (GFAP) and nerve growth related factor (GAP-43) in astrocytes and the changes of GFAP and GAP-43mRNA were detected by RealTime PCR method, and the relationship between the changes of these indexes and the exposure dose of NP was analyzed.
Results: (1) the effect of NP on serum hormone levels in weaning rats: the results of radioimmunoassay showed that the serum testosterone level decreased with the increase of NP exposure dose 21 days after birth, and there was a dose effect relationship (r=-0.889, P 0.05). Compared with the control group, the testosterone levels in two groups of M and H were significantly decreased (P0.05); estrogen level and NP exposure were also exposed. The dose positive correlation (r=-0.462, P0.01), the level of estrogen in the H dose group was significantly different from that of the control group (P 0.05). (2) the effect of NP exposure on the apoptosis of the nerve cells in the weaning and mature rats: the results of TUNEL assay showed that the apoptosis rate of hippocampal cells in 21d and 60d in the offspring was significantly higher than that in the control group (P 0.01), and the NP was NP. The exposure dose was positively correlated with the apoptosis rate (r=0.836,0.521, P 0.05). From the longitudinal comparison, the apoptosis rate of hippocampus tissue in the 21 day offspring of the exposed groups was higher than that of 60 days (t=3.331, P0.05). The effect of A. NP on the ChAT and AchE activity of the hippocampal tissues of the offspring: M, H dose group was significantly lower than that of the hippocampus. The activity of AchE in the M dose group was higher than that in the control group (P 0.01). The activity of ChAT and AchE in the hippocampus and the exposure dose of NP were in a dose-dependent manner (r=-0.821,0.757, P 0.05) and the expression of c-fos and c-jun protein in the.B. hippocampus tissue: the expression of c-jun and protein in the hippocampus of normal rats was very low, and the number of positive cells in the dose group was less and the pigment was shallow. The number of c-jun and c-fos positive cells in the hippocampus of the rats increased, especially in the high dose group. The effect of NP on the glial fibrillary acidic protein in the astrocytes of the hippocampus of the offspring: the results of the immunohistochemical technique for the expression of GFAP protein showed that the H dose group was 21d and the hippocampal and cortex of 60d old mice were GFAP free. The number of pestilence positive cells, the integral light density was significantly higher than that of the control group (P 0.05). The results of GFAP expression by fluorescence quantitative PCR showed that the expression of GFAP mRNA in the hippocampus of 21d and 60d age rats in the H dose group was significantly higher than that of the control group, and the expression of.GFAP protein and GFAP mRNA was positively related to the exposure dose of NP (0.05). The effect of NP on the nerve growth related protein in the offspring: the results of the immunohistochemical technique to detect the expression of GAP-43 protein showed that the average number of GAP-43 positive cells in the hippocampus and cortex of the H dose group was 21d and 60d, and the integral light density was lower than that of the control group (P 0.05), and the fluorescence quantitative PCR results showed 21d and 60d in the H dose group. The expression of GAP-43mRNA in hippocampus of aged rats was decreased, and the difference was statistically significant. There was a negative correlation between.GAP-43 protein and GAP-43mRNA expression and NP exposure dose (P 0.05).
Conclusion: combined with the above data, it is speculated that the possible mechanism of NP induced neurotoxicity is to contact NP in embryo and lactation period. The weak estrogen like action of NP causes the imbalance of endocrine system through competitive estrogen receptor, which leads to the increase of estrogen level in the offspring, changes the endocrine environment of the development of the offspring's brain and interferes with the hair cells. It reduces the expression level of neurotrophic factor GAP-43 and inhibits the differentiation of nerve cells, including neurite growth and branching, synapse formation, and increases the expression of glial fibrillary acidic protein GFAP, and affects the morphology, structure and energy of astrocytes, and further affects the cholinergic neurotransmitter Ach and the early basis. The information transmission of c-jun, c-fos and the induction of neuronal apoptosis in the hippocampus of the offspring led to the delay of the neural reflex time in the offspring's development and the learning and memory impairment at the mature stage.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R114

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