低剂量BDE-209单独和与BDE-47联合暴露对体外培养海马神经干细胞形态学及蛋白质组学的影响

发布时间：2018-04-11 13:06

本文选题：BDE-209 + BDE-47　；参考：《广州医学院》2012年硕士论文

【摘要】：【背景】 1.多溴联苯醚（Polybrominated Diphenyl Ethers，PBDEs）多溴联苯醚(Polybrominated Diphenyl Ethers，PBDEs)是一类溴代化合物，由于阻燃效率高、成本较低常作为阻燃剂添加到树脂、油漆、聚氨酯泡沫和聚苯乙烯等高分子合成材料中，广泛地应用于纺织品、塑料制品、电子电器和建筑材料等领域。早在1960年开始生产和使用多溴联苯醚，直到1981年瑞典发现多溴联苯醚是一种环境污染物。多溴联苯醚现已成为全球环境中普遍存在的污染物。有资料显示,大概80%的电子洋垃圾被运送到中国、印度等亚洲国家，而运送到中国的就占了90%。由于多溴联苯醚和多氯联苯醚（PCBs）具有相似的特征：难降解性、环境稳定性、高脂溶性、远距离迁移性和生物放大效应，能通过食物链传播，通过食物、大气、母乳和室内灰尘等在人体内蓄积，最终可危害人类的健康。PBDEs作用的主要靶器官是神经系统、生殖系统、脂肪组织和甲状腺等。动物实验证实多溴联苯醚有神经发育毒性、生殖毒性、免疫毒性和内分泌干扰毒性及潜在的致癌性。因此,在欧美等国家已限制生产和使用低溴类PBDEs，但一些高溴类PBDEs，如BDE-209因其阻燃性好，生物毒性不确切而仍被广泛使用。目前人们对PBDEs毒性的了解还远不如PCBs。实验室研究证据积累相对较多，而人群研究的对象主要是职业人群，数据相对缺乏。职业不同的人群中PBDEs同系物的分布也不相同。对中国、荷兰、美国及瑞士人群调查发现，非职业暴露人群体内主要为BDE-153，职业暴露人群体内主要为BDE-209和BDE-183。在现实环境中，多种污染物是同时存在，同时起作用。 2.十溴联苯醚（BDE-209）及四溴联苯醚（BDE-47）十溴联苯醚（BDE-209）是一种含有十个溴原子的多溴联苯醚，具有稳定性好，添加量少，价格便宜等优势，我国不仅是PBDEs的生产、使用和出口大国，而且还是接收电子垃圾的大国，其中BDE-209的生产使用量最大，因此BDE-209已成为我国重要的环境污染物。BDE-209在生产、使用和废物处理过程中通过一系列迁移转化进入沉积物、大气、生物固体及生物体中。进入生物体后，十溴联苯醚可代谢为低溴联苯醚、多溴二苯并呋喃及溴二苯并二VA英，引起更强的毒性效应。研究证明BDE-209具有神经发育毒性、肝脏毒性、甲状腺毒性、生殖毒性以及致癌性；母体内的BDE-209也可通过胎盘及乳汁传递给胎儿。但也有学者认为BDE-209在体内的代谢快，蓄积低，在常规暴露水平下对机体不造成影响。鉴于此，对于BDE-209的生物毒性的进一步研究是很有必要的。四溴联苯醚（BDE-47）也是PBDEs的主要同系物之一，与BDE-209同是近年环境中含量增长较快的持久性环境有机污染物。有研究报道示珠三角土壤中PBDEs污染严重，含量较高主要是BDE-209，BDE-47次之。由于BDE-209作为高溴化合物在体内代谢时可转化为BDE-47，转化过程中二者比例可发生不同变化。也有研究发现，BDE-209的作业人群体内存在高含量的BDE-209和相对较低含量BDE-47，停止暴露三年后，血样本中BDE-209的含量显著下降，但BDE-47含量并未发生明显改变，这一变化对生物体影响正是我们要关注的焦点。因此，开展BDE-209与BDE-47联合暴露对生物体影响的研究十分必要。 3.神经干细胞(neural stem cell， NSCs) 神经干细胞(NSCs)是干细胞的一种，，是存在于脑和脊髓中未分化的细胞，与其他干细胞一样，神经干细胞也具有自我更新、分裂增殖及多样分化的特点，它能够分化为神经元、星形胶质细胞和少突胶质细胞等多种类型的神经细胞，属于专能干细胞。神经干细胞的发现，为中枢神经性的重建和神经的再生提供了一个新的思路，为学习记忆机制的研究提供了一个新的视点。在哺乳动物胚胎期，神经干细胞主要分布在海马、嗅球、小脑、大脑皮质、脑室下区和侧脑室(室管膜上皮)；在成年后主要存在于海马、纹状体、脑室区、脑室下区及嗅球等部位。目前，NSCs研究的核心问题是NSCs增殖和分化的调控，研究表明，NSCs的增殖及分化的调控受多种因素的影响，有外源性因素(细胞因子和微环境)和内源性因素(基因)共同调控，其中起着决定作用的是内源性因素。个体发育是由干细胞不断分化形成功能细胞的过程，而神经系统发育也是胚胎干细胞不断分化成神经干细胞、再分化成神经细胞的过程，这分化过程任何一环节受影响均可导致神经元数目减少，从而引起神经功能缺陷。脑是由神经干细胞经过不断增殖、分化而最终形成。神经干细胞通过对称分裂进行自我更新，维持细胞种群，通过不对称分裂完成细胞分化，而外来化学物能够影响神经干细胞的自我更新潜能和发育分化的方向选择。 4.蛋白质组学（Proteomics）蛋白质组(Proteome)是指一种细胞或一个组织基因组所表达的全部蛋白质。蛋白质组学（Proteomics）是以基因组编码的所有蛋白质为研究对象，从细胞水平及整体水平研究蛋白质的组成及其变化规律，从而深入认识有机体的多种病理生理过程。蛋白质组学一词源于蛋白质（protein）与基因组学（genomics）两个词的组合，意指“一种基因组所表达的全套蛋白质”，即包括一种细胞乃至一种生物所表达的全部蛋白质。蛋白质组本质上指的是在大规模水平上研究蛋白质的特征，包括蛋白质的表达水平，翻译后的修饰，蛋白与蛋白相互作用等，由此获得蛋白质水平上的关于疾病发生，细胞代谢等过程的整体而全面的认识，这个概念最早是由Marc Wilkins在1995年提出的。因此，蛋白质组学研究不仅是探索生命奥秘的必须工作，也能为人类健康事业带来巨大的利益。蛋白质组学的研究是生命进入后基因时代的特征。由于NSCs分化过程中有众多的蛋白质、细胞因子的参与和消失,探索其功能和机制的过程是极其复杂和庞大的,而蛋白质组学具有规模大和高通量等优点,与基因组学、生物信息学互相渗透、互相补充。PBDEs可影响NSCs的增殖和分化，但是其影响机制尚未完全阐明，因而本课题从细胞水平,给予体外培养神经干细胞低剂量BDE-209及BDE-47联合暴露，研究BDE-209及BDE-47联合暴露对神经干细形态学及蛋白质组学的影响，以更全面地了解BDE-209及BDE-47神经发育毒性的影响关键点，更深入地了解PBDEs与先天神经发育异常的关系，为临床上对PBDEs引起的神经发育异常进行有效地检测和预防提供理论依据。本课题分为以下两个部分进行各项具体实验。第一部分低剂量BDE-209单独和与BDE-47联合暴露对体外培养神经干细胞的形态学的影响【目的】取24小时内新生SD大鼠的海马组织进行无血清传代培养，对神经干细胞进行鉴定和纯度检测，染毒后分别测量形成神经球数目及神经球直径，观察BDE-209单独和与BDE-47联合暴露对体外培养新生鼠海马神经干细胞形态的影响。【材料与方法】 1.购买清洁级24小时内新生SD大鼠，取大鼠海马组织进行体外神经干细胞的培养。 2.对培养3-4代后的海马神经干细胞进行鉴定及纯度检测。 3.将传代培养3-4代的新生鼠海马神经干细胞吹打成单细胞悬液，之后进行BDE-209及BDE-47染毒，实验共分8组：对照组（DMSO），0.6ug/ml BDE-209组，1.0ug/ml BDE-209组，6.0ug/ml BDE-209组，3.2ug/ml BDE-47组，0.6ug/mlBDE-209+3.2ug/ml BDE-47组，1.0ug/ml BDE-209+3.2ug/ml BDE-47组，6.0ug/ml BDE-209+3.2ug/ml BDE-47组，每组设5个平行样。对照组加入含1‰DMSO的培养液。 4.染毒72h后，在倒置显微镜下进行神经干细胞形态观察，利用图象分析软件记录形成神经球的个数，测量神经球的平均直径。【结果】 1.神经干细胞鉴定：海马组织经培养3～5天可见神经球形成,之后可见神经球逐渐增大，经免疫细胞化学染色，神经干细胞的标记性蛋白巢蛋白(Nestin)表达阳性，证实所分离培养的细胞是神经干细胞。 2.海马神经干细胞培养3～4代后在倒置显微镜下观察，由数十到数百个细胞聚集形成大小不等的神经球，表面可见单细胞突出呈鱼泡状，折光性强，周围光晕明显，吹打成单细胞悬液后行免疫细胞化学法对神经干细胞的纯度进行检测。由结果可见分散的神经干细胞占主体,可占90%以上。 3.利用图象分析软件记录形成神经球的个数，测量神经球的平均直径，发现低剂量BDE-209染毒组(0.6ug/ml BDE-209组)形成神经球个数与对照组无显著差异。随着染毒剂量的增加，神经球数目变少。还发现低剂量BDE-209染毒组(0.6ug/ml BDE-209组和1.0ug/ml BDE-209)形成神经球直径大小与对照组无显著差异。BDE-209和BDE-47联合染毒对形成神经球个数的影响存在交互作用(P0．05)。【结论】以上提示：无血清培养的新生鼠海马组织神经干细胞具有自我更新和增殖能力，神经干细胞形成悬浮生长的神经球，经免疫细胞化学鉴定所培养的是神经干细胞。一定剂量的BDE-209和BDE-47可导致新生鼠海马神经干细胞形成神经球数目及直径改变，BDE-209与BDE-47之间对神经干细胞的生长存在一定的交互作用。第二部分低剂量BDE-209单独和与BDE-47联合暴露对体外培养神经干细胞蛋白组学的影响【目的】采用蛋白质组学研究BDE-209及BDE-47对神经干细胞蛋白的影响，从蛋白质水平探讨PBDES神经发育毒性机制。【材料与方法】 1.传代培养3-4代的新生鼠海马神经干细胞暴露于BDE-209及BDE-47，实验共分4组：（1）对照组（DMSO）；（2）6.0ug/ml BDE-209组；（3）3.2ug/mlBDE-47组；（4）6.0ug/ml BDE-209+3.2ug/mlBDE-47组。每组设5个平行样。对照组加入含1‰DMSO的培养液。 2.染毒培养72h后，离心收集细胞，提取各组总蛋白质，采用Bradford法测定蛋白质样品浓度。 3.进行双向凝胶电泳（2-DE）分离差异蛋白：第一向等电聚焦（IEF）；第二向SDS-PAGE. 4.凝胶染色：分析胶银染，质谱胶考染。 5.银染后采用Powerlook1100扫描仪对染色后的双向凝胶电泳胶进行扫描获取图像，采用Image Master2D platinum5.0凝胶图像分析软件进行分析识别差异表达的蛋白质点。 6.对表达差异1.8倍以上蛋白质点进行胶内酶解和MALDI-TOF-MS检测，获得肽质量指纹谱，应用BioTools搜索软件在NCBI数据库中进行检索鉴定。 7.应用Western blot对其中2个蛋白质进行验证。【结果】建立了暴露于BDE-209及BDE-47的神经干细胞蛋白质的2-DE图谱；识别了39个差异表达的蛋白质点，质谱鉴定去冗余后确定了19种蛋白质；Westernblot验证了丝切蛋白l（cofilin-1）和波形蛋白（vimentin）的差异表达水平，结果与双向电泳结果一致。【结论】 BDE-209和BDE-47可引起神经干细胞蛋白质表达谱的改变，应用MALDI-TOF-MS初步鉴定出了19种蛋白质，19个差异表达蛋白质为研究PBDEs神经发育毒性机制提供了实验依据，这为深入研究PBDEs神经毒性机制打下了基础。
[Abstract]:[background]
1. polybrominated diphenyl ethers (Polybrominated Diphenyl Ethers, PBDEs)
Polybrominated diphenyl ethers (Polybrominated Diphenyl, Ethers, PBDEs) is a class of brominated compounds as flame retardant high efficiency, low cost is often used as a flame retardant is added to the resin, paint, polyurethane foam and polystyrene polymer materials, widely used in textiles, plastic products, electronic appliances and building materials such as early. In 1960 the production and use of PBDEs, until 1981 in Sweden found PBDEs is an environmental pollutant. PBDEs have become ubiquitous environmental pollutants globally. Data shows that about 80% of the electronic garbage is transported to Chinese, India and other Asian countries, and transported to the China accounted for 90%.
Because of polybrominated diphenyl ethers and polychlorinated diphenyl ethers (PCBs) have similar characteristics: refractory, environmental stability, high liposolubility, long-distance migration and biological amplification, can be transmitted through the food chain, through food, air, milk and indoor dust accumulation in the body and so on, the main target organ health.PBDEs final can be harmful to human's nervous system, reproductive system, adipose tissue and thyroid. Animal experiments confirmed that PBDEs are toxic, neural development, reproductive toxicity, immune toxicity and endocrine toxicity and potential carcinogenicity. Therefore, in Europe and other countries have restricted the production and use of low bromine PBDEs, but some high br class PBDEs, such as BDE-209 because of its good flame retardancy, biological toxicity is not exact and is still widely used. The current understanding of the toxicity of PBDEs is far better than PCBs. laboratory evidence of accumulation of more, and the crowd of The main target is the occupation population, the relative lack of data. The distribution of occupation different populations of PBDEs congeners are not the same. For Chinese, Holland, the United States and the Swiss population survey found, non occupation exposure in vivo is mainly BDE-153, occupation exposure population in vivo is mainly BDE-209 and BDE-183. in the real environment, a variety of pollutants exist at the same time at the same time, play a role.
20 brominated diphenyl ethers (BDE-209) and four brominated diphenyl ethers (BDE-47)
Ten polybrominated diphenyl ether (BDE-209) is a bromine atom containing ten polybrominated diphenyl ethers, with good stability, add less, low price advantage, China is not only PBDEs production, use and export country, and still receive electronic waste power, which BDE-209 production is the largest, therefore BDE-209 has become China's important environmental pollutants.BDE-209 in the production, use and waste treatment process through a series of migration and transformation into sediment, atmosphere, biological solids and organisms. Enter the organisms, ten polybrominated diphenyl ether can be metabolized to low brominated diphenyl ether, two polybrominated dibenzofuran and two bromo benzene and two VA Britain, caused by toxic effects. The study shows that BDE-209 has stronger toxicity, neural development toxicity for liver, thyroid toxicity, reproductive toxicity and carcinogenicity; maternal BDE-209 can pass through the placenta and milk but also learn the fetus. It is believed that the metabolism of BDE-209 is fast and low in the body, and it does not affect the organism at regular exposure level. In view of this, further research on the biological toxicity of BDE-209 is necessary.
Four polybrominated diphenyl ether (BDE-47) is one of the main homologues PBDEs and BDE-209, with the rapid growth in recent years the content of environment environmental persistent organic pollutants. Studies have reported PBDEs in the Pearl River Delta soil pollution, high content is mainly BDE-209, BDE-47. The BDE-209 as high brominated compounds can be metabolized in the body into BDE-47, ratio of the two conversion process can be changed. Research has also found that the presence of high content of BDE-209 and the relatively low content of BDE-47 BDE-209 workers in three years after stopping exposure, the content of BDE-209 in blood samples was significantly decreased, but the content of BDE-47 did not change, the effect of this change we want to focus on organisms is concerned. Therefore, the research of BDE-209 and BDE-47 to carry out joint exposure to the organism effect is necessary.
3. neural stem cells (neural stem cell, NSCs)
Neural stem cells (NSCs) is a kind of stem cells, is present in undifferentiated cells in the brain and spinal cord, like other stem cells, neural stem cells have self-renewal, proliferation and differentiation characteristics of diversity, it can differentiate into neurons, astrocytes and various types of oligodendrocytes. Nerve cells are multipotent neural stem cells. The discovery provides a new way for the regeneration of central nerve and nerve reconstruction, provides a new perspective for the study of learning and memory mechanism. In mammalian embryos, neural stem cells were mainly distributed in the hippocampus, olfactory bulb, cerebellum the cerebral cortex, subventricular zone and the lateral ventricle (ependymal epithelium); in the adult mainly exists in the striatum, hippocampus, subventricular zone, subventricular zone and olfactory bulb and other parts. At present, the core problem of NSCs research is to regulate the proliferation and differentiation of NSCs research. The regulation of proliferation and differentiation of NSCs is influenced by many factors, including exogenous factors (cytokines and microenvironment) and endogenous factors (genes), which play a decisive role in endogenous factors.
Ontogeny is a stem cell differentiation and formation process of the cell, and the development of the nervous system is embryonic stem cells differentiation into neural stem cells, and then differentiate into nerve cells, the differentiation of any link affected can lead to the reduction in the number of neurons, causing neurological defects by neural stem cells after cerebral continuous proliferation, differentiation of neural stem cells and eventually formed. By symmetrically to self renew and maintain cell populations by asymmetric division to complete cell differentiation, and xenobiotics can self renew selection potential and the development and differentiation of neural stem cells. The influence of direction
4. proteomics (Proteomics)
Protein group (Proteome) refers to all proteins a cell or a tissue expressed by genome. Proteomics (Proteomics) is a protein encoding all genome as the research object, from the composition and the law of changes in the cellular level and the overall level of protein, thus further understanding the pathophysiological process of multiple protein organisms. Group learning comes from the protein (protein) and genomics (genomics) a combination of two words, means the whole set of proteins expressed by a genome, including a cell or an organism protein expression. The protein group essentially refers to the characteristics of the protein on a large scale level, including the level of protein expression, post-translational modification, protein and protein interaction, thus obtained the protein level on the occurrence of disease, cell metabolism of the whole Body and comprehensive understanding, this concept was first proposed by Marc Wilkins in 1995. Therefore, proteomics research is not only to explore the mystery of life must work, also can bring huge benefits to human health. The study of proteomics is characteristic of life after entering the gene generation.
Because there are many protein NSCs in the differentiation process of cytokines and disappear, to explore the function and mechanism of the process is extremely complex and huge, and proteomics has the advantages of large scale and high throughput, and genomics, bioinformatics, mutual penetration, mutual supplement of.PBDEs can influence the proliferation and differentiation of NSCs however, the influence mechanism has not been fully elucidated, therefore the issue from the cellular level to neural stem cells and BDE-47 combined with low dose BDE-209 exposure in vitro study of BDE-209 and BDE-47 combined with exposure to the effects of neural stem morphologic and proteomics, to more fully understand the toxic effects of BDE-209 and BDE-47 neural development key point and a deeper understanding of the relationship between abnormal PBDEs and congenital neural development, as abnormalities of PBDEs in clinical nerve to effectively detect and provide a theoretical basis for the prevention. The subject is divided into the following two parts to carry out specific experiments.
Part one
The effect of low dose BDE-209 alone and combined with BDE-47 exposure on the morphology of cultured neural stem cells in vitro
[Objective] from neonatal SD rats within 24 hours of the hippocampus by serum-free culture, identification and purity detection of neural stem cells were measured after exposure to form neurospheres and number of neural ball diameter, observation of BDE-209 alone and in combination with BDE-47 exposure on hippocampus nerve stem cells in vitro.
[materials and methods]
1. the newborn SD rats were purchased for 24 hours in the clean grade, and the rat hippocampus tissue was cultured for the culture of neural stem cells in vitro.
2. the identification and purity test of hippocampal neural stem cells were carried out after 3-4 generations of culture.
3. will be the 3-4 generation of neonatal rat hippocampal neural stem cells into monoplast suspension culture, after BDE-209 and BDE-47 exposure, the subjects were divided into 8 groups: control group (DMSO), 0.6ug/ml BDE-209 group, 1.0ug/ml BDE-209 group, 6.0ug/ml BDE-209 group, 3.2ug/ml BDE-47 group, 0.6ug/ mlBDE-209+3.2ug/ml group BDE-47 BDE-209+3.2ug/ml group BDE-47, 1.0ug/ml 6.0ug/ml, BDE-209+3.2ug/ml BDE-47 group, each group had 5 parallel samples. The control group culture medium containing 1 DMSO%.
4. after exposure to 72h, the morphology of neural stem cells was observed under inverted microscope. The number of neurospheres was recorded by image analysis software, and the mean diameter of nerve spheres was measured.
[results]
1.: neural stem cells identified in the hippocampus were cultured for 3~5 days after the formation of neurospheres visible, visible neurospheres gradually increased by immunocytochemical staining, neural stem cell marker nestin protein (Nestin) expression, confirmed that the cultured cells of neural stem cells.
Cell culture after 3~4 generations observed under inverted microscope 2. in hippocampus from tens to hundreds of cell aggregation sizes of neurospheres, visible on the surface of single cell was prominent fish bubble, strong refraction, obvious halo around, into monoplast suspension by immunocytochemical method of neural stem cells the purity was detected. Results from the dispersion of neural stem cells can be dominant, accounting for more than 90%.
3. by using the image analysis software to record the number of neurospheres formed, the average measuring diameter of neurospheres, low dose BDE-209 treated group (0.6ug/ml group BDE-209) formed neurospheres number had no significant difference compared with the control group. With the increase of the dose, number of neurospheres decreased. We also found low dose exposure group (BDE-209 0.6ug/ml BDE-209 group and 1.0ug/ml BDE-209) to form neurospheres diameter had no significant difference with control group.BDE-209 and BDE-47 combined interaction influence on the formation of a number of neurospheres (P0.05).
[Conclusion]
These results suggest that neonatal rat hippocampus neural stem cells in serum-free medium have the ability of self-renewal and proliferation of neural stem cells, and formed neurospheres, by immunocytochemical identification of cultured neural stem cells. A certain dose of BDE-209 and BDE-47 can lead to new neural stem cells of rat hippocampal formation of neurospheres and the number of the diameter change between BDE-209 and BDE-47 on neural stem cells have certain effects.
The second part
Effects of low dose BDE-209 alone and combined with BDE-47 exposure on the in vitro cultured neural stem cell proteomics
[Objective]
The effects of BDE-209 and BDE-47 on the protein of neural stem cells were studied by proteomics, and the mechanism of PBDES neurodevelopmental toxicity was discussed from protein level.
[materials and methods]
The 3-4 generation of neonatal rat hippocampal neural stem cells were exposed to BDE-209 and BDE-47 cultured for 1. passages, the subjects were divided into 4 groups: (1) control group (DMSO); (2) 6.0ug/ml (3) BDE-209 group; 3.2ug/mlBDE-47 group; (4) 6.0ug/ml BDE-209+3.2ug/mlBDE-47 group. Each group had 5 parallel samples of culture medium as. Group containing 1 DMSO%.
After 2. exposure to 72h, the cells were collected by centrifugation and the total protein was extracted. The concentration of protein samples was measured by Bradford.
3. bi-directional gel electrophoresis (gels).

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

【参考文献】