本文選題：BDE-209 + BDE-47��； 參考：《廣州醫(yī)學(xué)院》2012年碩士論文

【摘要】：【背景】 1.多溴聯(lián)苯醚（Polybrominated Diphenyl Ethers，PBDEs） 多溴聯(lián)苯醚(Polybrominated Diphenyl Ethers，PBDEs)是一類溴代化合物，由于阻燃效率高、成本較低常作為阻燃劑添加到樹脂、油漆、聚氨酯泡沫和聚苯乙烯等高分子合成材料中，廣泛地應(yīng)用于紡織品、塑料制品、電子電器和建筑材料等領(lǐng)域。早在1960年開始生產(chǎn)和使用多溴聯(lián)苯醚，直到1981年瑞典發(fā)現(xiàn)多溴聯(lián)苯醚是一種環(huán)境污染物。多溴聯(lián)苯醚現(xiàn)已成為全球環(huán)境中普遍存在的污染物。有資料顯示,大概80%的電子洋垃圾被運(yùn)送到中國、印度等亞洲國家，而運(yùn)送到中國的就占了90%。 由于多溴聯(lián)苯醚和多氯聯(lián)苯醚（PCBs）具有相似的特征：難降解性、環(huán)境穩(wěn)定性、高脂溶性、遠(yuǎn)距離遷移性和生物放大效應(yīng)，能通過食物鏈傳播，通過食物、大氣、母乳和室內(nèi)灰塵等在人體內(nèi)蓄積，最終可危害人類的健康。PBDEs作用的主要靶器官是神經(jīng)系統(tǒng)、生殖系統(tǒng)、脂肪組織和甲狀腺等。動物實(shí)驗(yàn)證實(shí)多溴聯(lián)苯醚有神經(jīng)發(fā)育毒性、生殖毒性、免疫毒性和內(nèi)分泌干擾毒性及潛在的致癌性。因此,在歐美等國家已限制生產(chǎn)和使用低溴類PBDEs，但一些高溴類PBDEs，如BDE-209因其阻燃性好，生物毒性不確切而仍被廣泛使用。目前人們對PBDEs毒性的了解還遠(yuǎn)不如PCBs。實(shí)驗(yàn)室研究證據(jù)積累相對較多，而人群研究的對象主要是職業(yè)人群，數(shù)據(jù)相對缺乏。職業(yè)不同的人群中PBDEs同系物的分布也不相同。對中國、荷蘭、美國及瑞士人群調(diào)查發(fā)現(xiàn)，非職業(yè)暴露人群體內(nèi)主要為BDE-153，職業(yè)暴露人群體內(nèi)主要為BDE-209和BDE-183。在現(xiàn)實(shí)環(huán)境中，多種污染物是同時(shí)存在，同時(shí)起作用。 2.十溴聯(lián)苯醚（BDE-209）及四溴聯(lián)苯醚（BDE-47） 十溴聯(lián)苯醚（BDE-209）是一種含有十個溴原子的多溴聯(lián)苯醚，具有穩(wěn)定性好，添加量少，價(jià)格便宜等優(yōu)勢，我國不僅是PBDEs的生產(chǎn)、使用和出口大國，而且還是接收電子垃圾的大國，其中BDE-209的生產(chǎn)使用量最大，因此BDE-209已成為我國重要的環(huán)境污染物。BDE-209在生產(chǎn)、使用和廢物處理過程中通過一系列遷移轉(zhuǎn)化進(jìn)入沉積物、大氣、生物固體及生物體中。進(jìn)入生物體后，十溴聯(lián)苯醚可代謝為低溴聯(lián)苯醚、多溴二苯并呋喃及溴二苯并二VA英，引起更強(qiáng)的毒性效應(yīng)。研究證明BDE-209具有神經(jīng)發(fā)育毒性、肝臟毒性、甲狀腺毒性、生殖毒性以及致癌性；母體內(nèi)的BDE-209也可通過胎盤及乳汁傳遞給胎兒。但也有學(xué)者認(rèn)為BDE-209在體內(nèi)的代謝快，蓄積低，在常規(guī)暴露水平下對機(jī)體不造成影響。鑒于此，對于BDE-209的生物毒性的進(jìn)一步研究是很有必要的。 四溴聯(lián)苯醚（BDE-47）也是PBDEs的主要同系物之一，與BDE-209同是近年環(huán)境中含量增長較快的持久性環(huán)境有機(jī)污染物。有研究報(bào)道示珠三角土壤中PBDEs污染嚴(yán)重，含量較高主要是BDE-209，BDE-47次之。由于BDE-209作為高溴化合物在體內(nèi)代謝時(shí)可轉(zhuǎn)化為BDE-47，轉(zhuǎn)化過程中二者比例可發(fā)生不同變化。也有研究發(fā)現(xiàn)，BDE-209的作業(yè)人群體內(nèi)存在高含量的BDE-209和相對較低含量BDE-47，停止暴露三年后，血樣本中BDE-209的含量顯著下降，但BDE-47含量并未發(fā)生明顯改變，這一變化對生物體影響正是我們要關(guān)注的焦點(diǎn)。因此，開展BDE-209與BDE-47聯(lián)合暴露對生物體影響的研究十分必要。 3.神經(jīng)干細(xì)胞(neural stem cell， NSCs) 神經(jīng)干細(xì)胞(NSCs)是干細(xì)胞的一種，，是存在于腦和脊髓中未分化的細(xì)胞，與其他干細(xì)胞一樣，神經(jīng)干細(xì)胞也具有自我更新、分裂增殖及多樣分化的特點(diǎn)，它能夠分化為神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞等多種類型的神經(jīng)細(xì)胞，屬于專能干細(xì)胞。神經(jīng)干細(xì)胞的發(fā)現(xiàn)，為中樞神經(jīng)性的重建和神經(jīng)的再生提供了一個新的思路，為學(xué)習(xí)記憶機(jī)制的研究提供了一個新的視點(diǎn)。在哺乳動物胚胎期，神經(jīng)干細(xì)胞主要分布在海馬、嗅球、小腦、大腦皮質(zhì)、腦室下區(qū)和側(cè)腦室(室管膜上皮)；在成年后主要存在于海馬、紋狀體、腦室區(qū)、腦室下區(qū)及嗅球等部位。目前，NSCs研究的核心問題是NSCs增殖和分化的調(diào)控，研究表明，NSCs的增殖及分化的調(diào)控受多種因素的影響，有外源性因素(細(xì)胞因子和微環(huán)境)和內(nèi)源性因素(基因)共同調(diào)控，其中起著決定作用的是內(nèi)源性因素。 個體發(fā)育是由干細(xì)胞不斷分化形成功能細(xì)胞的過程，而神經(jīng)系統(tǒng)發(fā)育也是胚胎干細(xì)胞不斷分化成神經(jīng)干細(xì)胞、再分化成神經(jīng)細(xì)胞的過程，這分化過程任何一環(huán)節(jié)受影響均可導(dǎo)致神經(jīng)元數(shù)目減少，從而引起神經(jīng)功能缺陷。腦是由神經(jīng)干細(xì)胞經(jīng)過不斷增殖、分化而最終形成。神經(jīng)干細(xì)胞通過對稱分裂進(jìn)行自我更新，維持細(xì)胞種群，通過不對稱分裂完成細(xì)胞分化，而外來化學(xué)物能夠影響神經(jīng)干細(xì)胞的自我更新潛能和發(fā)育分化的方向選擇。 4.蛋白質(zhì)組學(xué)（Proteomics） 蛋白質(zhì)組(Proteome)是指一種細(xì)胞或一個組織基因組所表達(dá)的全部蛋白質(zhì)。蛋白質(zhì)組學(xué)（Proteomics）是以基因組編碼的所有蛋白質(zhì)為研究對象，從細(xì)胞水平及整體水平研究蛋白質(zhì)的組成及其變化規(guī)律，從而深入認(rèn)識有機(jī)體的多種病理生理過程。蛋白質(zhì)組學(xué)一詞源于蛋白質(zhì)（protein）與基因組學(xué)（genomics）兩個詞的組合，意指“一種基因組所表達(dá)的全套蛋白質(zhì)”，即包括一種細(xì)胞乃至一種生物所表達(dá)的全部蛋白質(zhì)。蛋白質(zhì)組本質(zhì)上指的是在大規(guī)模水平上研究蛋白質(zhì)的特征，包括蛋白質(zhì)的表達(dá)水平，翻譯后的修飾，蛋白與蛋白相互作用等，由此獲得蛋白質(zhì)水平上的關(guān)于疾病發(fā)生，細(xì)胞代謝等過程的整體而全面的認(rèn)識，這個概念最早是由Marc Wilkins在1995年提出的。因此，蛋白質(zhì)組學(xué)研究不僅是探索生命奧秘的必須工作，也能為人類健康事業(yè)帶來巨大的利益。蛋白質(zhì)組學(xué)的研究是生命進(jìn)入后基因時(shí)代的特征。 由于NSCs分化過程中有眾多的蛋白質(zhì)、細(xì)胞因子的參與和消失,探索其功能和機(jī)制的過程是極其復(fù)雜和龐大的,而蛋白質(zhì)組學(xué)具有規(guī)模大和高通量等優(yōu)點(diǎn),與基因組學(xué)、生物信息學(xué)互相滲透、互相補(bǔ)充。PBDEs可影響NSCs的增殖和分化，但是其影響機(jī)制尚未完全闡明，因而本課題從細(xì)胞水平,給予體外培養(yǎng)神經(jīng)干細(xì)胞低劑量BDE-209及BDE-47聯(lián)合暴露，研究BDE-209及BDE-47聯(lián)合暴露對神經(jīng)干細(xì)形態(tài)學(xué)及蛋白質(zhì)組學(xué)的影響，以更全面地了解BDE-209及BDE-47神經(jīng)發(fā)育毒性的影響關(guān)鍵點(diǎn)，更深入地了解PBDEs與先天神經(jīng)發(fā)育異常的關(guān)系，為臨床上對PBDEs引起的神經(jīng)發(fā)育異常進(jìn)行有效地檢測和預(yù)防提供理論依據(jù)。本課題分為以下兩個部分進(jìn)行各項(xiàng)具體實(shí)驗(yàn)。 第一部分 低劑量BDE-209單獨(dú)和與BDE-47聯(lián)合暴露對體外培養(yǎng)神經(jīng)干細(xì)胞的形態(tài)學(xué)的影響 【目的】取24小時(shí)內(nèi)新生SD大鼠的海馬組織進(jìn)行無血清傳代培養(yǎng)，對神經(jīng)干細(xì)胞進(jìn)行鑒定和純度檢測，染毒后分別測量形成神經(jīng)球數(shù)目及神經(jīng)球直徑，觀察BDE-209單獨(dú)和與BDE-47聯(lián)合暴露對體外培養(yǎng)新生鼠海馬神經(jīng)干細(xì)胞形態(tài)的影響。 【材料與方法】 1.購買清潔級24小時(shí)內(nèi)新生SD大鼠，取大鼠海馬組織進(jìn)行體外神經(jīng)干細(xì)胞的培養(yǎng)。 2.對培養(yǎng)3-4代后的海馬神經(jīng)干細(xì)胞進(jìn)行鑒定及純度檢測。 3.將傳代培養(yǎng)3-4代的新生鼠海馬神經(jīng)干細(xì)胞吹打成單細(xì)胞懸液，之后進(jìn)行BDE-209及BDE-47染毒，實(shí)驗(yàn)共分8組：對照組（DMSO），0.6ug/ml BDE-209組，1.0ug/ml BDE-209組，6.0ug/ml BDE-209組，3.2ug/ml BDE-47組，0.6ug/mlBDE-209+3.2ug/ml BDE-47組，1.0ug/ml BDE-209+3.2ug/ml BDE-47組，6.0ug/ml BDE-209+3.2ug/ml BDE-47組，每組設(shè)5個平行樣。對照組加入含1‰DMSO的培養(yǎng)液。 4.染毒72h后，在倒置顯微鏡下進(jìn)行神經(jīng)干細(xì)胞形態(tài)觀察，利用圖象分析軟件記錄形成神經(jīng)球的個數(shù)，測量神經(jīng)球的平均直徑。 【結(jié)果】 1.神經(jīng)干細(xì)胞鑒定：海馬組織經(jīng)培養(yǎng)3～5天可見神經(jīng)球形成,之后可見神經(jīng)球逐漸增大，經(jīng)免疫細(xì)胞化學(xué)染色，神經(jīng)干細(xì)胞的標(biāo)記性蛋白巢蛋白(Nestin)表達(dá)陽性，證實(shí)所分離培養(yǎng)的細(xì)胞是神經(jīng)干細(xì)胞。 2.海馬神經(jīng)干細(xì)胞培養(yǎng)3～4代后在倒置顯微鏡下觀察，由數(shù)十到數(shù)百個細(xì)胞聚集形成大小不等的神經(jīng)球，表面可見單細(xì)胞突出呈魚泡狀，折光性強(qiáng)，周圍光暈明顯，吹打成單細(xì)胞懸液后行免疫細(xì)胞化學(xué)法對神經(jīng)干細(xì)胞的純度進(jìn)行檢測。由結(jié)果可見分散的神經(jīng)干細(xì)胞占主體,可占90%以上。 3.利用圖象分析軟件記錄形成神經(jīng)球的個數(shù)，測量神經(jīng)球的平均直徑，發(fā)現(xiàn)低劑量BDE-209染毒組(0.6ug/ml BDE-209組)形成神經(jīng)球個數(shù)與對照組無顯著差異。隨著染毒劑量的增加，神經(jīng)球數(shù)目變少。還發(fā)現(xiàn)低劑量BDE-209染毒組(0.6ug/ml BDE-209組和1.0ug/ml BDE-209)形成神經(jīng)球直徑大小與對照組無顯著差異。BDE-209和BDE-47聯(lián)合染毒對形成神經(jīng)球個數(shù)的影響存在交互作用(P0．05)。 【結(jié)論】 以上提示：無血清培養(yǎng)的新生鼠海馬組織神經(jīng)干細(xì)胞具有自我更新和增殖能力，神經(jīng)干細(xì)胞形成懸浮生長的神經(jīng)球，經(jīng)免疫細(xì)胞化學(xué)鑒定所培養(yǎng)的是神經(jīng)干細(xì)胞。一定劑量的BDE-209和BDE-47可導(dǎo)致新生鼠海馬神經(jīng)干細(xì)胞形成神經(jīng)球數(shù)目及直徑改變，BDE-209與BDE-47之間對神經(jīng)干細(xì)胞的生長存在一定的交互作用。 第二部分 低劑量BDE-209單獨(dú)和與BDE-47聯(lián)合暴露對體外培養(yǎng)神經(jīng)干細(xì)胞蛋白組學(xué)的影響 【目的】 采用蛋白質(zhì)組學(xué)研究BDE-209及BDE-47對神經(jīng)干細(xì)胞蛋白的影響，從蛋白質(zhì)水平探討PBDES神經(jīng)發(fā)育毒性機(jī)制。 【材料與方法】 1.傳代培養(yǎng)3-4代的新生鼠海馬神經(jīng)干細(xì)胞暴露于BDE-209及BDE-47，實(shí)驗(yàn)共分4組：（1）對照組（DMSO）；（2）6.0ug/ml BDE-209組；（3）3.2ug/mlBDE-47組；（4）6.0ug/ml BDE-209+3.2ug/mlBDE-47組。每組設(shè)5個平行樣。對照組加入含1‰DMSO的培養(yǎng)液。 2.染毒培養(yǎng)72h后，離心收集細(xì)胞，提取各組總蛋白質(zhì)，采用Bradford法測定蛋白質(zhì)樣品濃度。 3.進(jìn)行雙向凝膠電泳（2-DE）分離差異蛋白：第一向等電聚焦（IEF）；第二向SDS-PAGE. 4.凝膠染色：分析膠銀染，質(zhì)譜膠考染。 5.銀染后采用Powerlook1100掃描儀對染色后的雙向凝膠電泳膠進(jìn)行掃描獲取圖像，采用Image Master2D platinum5.0凝膠圖像分析軟件進(jìn)行分析識別差異表達(dá)的蛋白質(zhì)點(diǎn)。 6.對表達(dá)差異1.8倍以上蛋白質(zhì)點(diǎn)進(jìn)行膠內(nèi)酶解和MALDI-TOF-MS檢測，獲得肽質(zhì)量指紋譜，應(yīng)用BioTools搜索軟件在NCBI數(shù)據(jù)庫中進(jìn)行檢索鑒定。 7.應(yīng)用Western blot對其中2個蛋白質(zhì)進(jìn)行驗(yàn)證。 【結(jié)果】 建立了暴露于BDE-209及BDE-47的神經(jīng)干細(xì)胞蛋白質(zhì)的2-DE圖譜；識別了39個差異表達(dá)的蛋白質(zhì)點(diǎn)，質(zhì)譜鑒定去冗余后確定了19種蛋白質(zhì)；Westernblot驗(yàn)證了絲切蛋白l（cofilin-1）和波形蛋白（vimentin）的差異表達(dá)水平，結(jié)果與雙向電泳結(jié)果一致。 【結(jié)論】 BDE-209和BDE-47可引起神經(jīng)干細(xì)胞蛋白質(zhì)表達(dá)譜的改變，應(yīng)用MALDI-TOF-MS初步鑒定出了19種蛋白質(zhì)，19個差異表達(dá)蛋白質(zhì)為研究PBDEs神經(jīng)發(fā)育毒性機(jī)制提供了實(shí)驗(yàn)依據(jù)，這為深入研究PBDEs神經(jīng)毒性機(jī)制打下了基礎(chǔ)。
[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).

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114

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