本文關(guān)鍵詞： 全氟辛烷磺酸 血—腦屏障 MAPK信號(hào)通路 ICR小鼠 星形膠質(zhì)細(xì)胞　出處：《南京醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：全氟辛烷磺酸/鹽（Perfluorooctane sulfonate，PFOS）是一類近年來引起廣泛關(guān)注的新型環(huán)境持久性有機(jī)污染物（Persist organic pollutants，POPs）。PFOS類化合物具有難降解性、生物蓄積性和食物鏈放大作用等，其所造成的環(huán)境污染已遍及全球生態(tài)系統(tǒng)，成為近年來學(xué)界的研究熱點(diǎn)。PFOS不但可誘導(dǎo)肝臟、免疫及內(nèi)分泌系統(tǒng)的損害，而且對(duì)動(dòng)物的神經(jīng)發(fā)育也有影響。近年來在PFOS神經(jīng)毒性研究中，主要關(guān)注了PFOS對(duì)各類神經(jīng)元細(xì)胞的作用以及經(jīng)此途徑誘導(dǎo)神經(jīng)損傷的相關(guān)分子機(jī)制。血—腦屏障是存在于腦和脊髓內(nèi)的毛細(xì)血管與神經(jīng)組織之間的一個(gè)調(diào)節(jié)中樞神經(jīng)系統(tǒng)內(nèi)環(huán)境的細(xì)胞屏障，能夠維持腦內(nèi)離子、激素和遞質(zhì)等的動(dòng)態(tài)平衡。血—腦屏障能夠被多種外源性因素所影響。目前研究較多的是重金屬、電離輻射和創(chuàng)傷等，外源性化學(xué)物對(duì)血—腦屏障的影響研究較少。PFOS若能夠?qū)ι窠?jīng)元細(xì)胞造成損傷，血—腦屏障可能是關(guān)鍵環(huán)節(jié)，因此，闡明PFOS對(duì)血—腦屏障的影響及其分子機(jī)制有助于全面認(rèn)識(shí)PFOS誘導(dǎo)的神經(jīng)損傷過程。 為此，我們利用ICR雄性小鼠作為PFOS的染毒模型，通過檢測(cè)腦中PFOS含量、觀察腦組織的形態(tài)學(xué)改變情況、緊密連接相關(guān)蛋白和損傷相關(guān)蛋白的表達(dá)情況以及血清中相關(guān)激素水平等，系統(tǒng)評(píng)估PFOS對(duì)血—腦屏障的結(jié)構(gòu)和功能的影響，尋找PFOS所致?lián)p傷效應(yīng)過程中的靶細(xì)胞和靶分子，并通過體外研究進(jìn)一步探討PFOS影響血—腦屏障的分子機(jī)制。為環(huán)境持久性有機(jī)污染物的危害評(píng)價(jià)、風(fēng)險(xiǎn)評(píng)估以及預(yù)防和控制策略制定提供重要參考。 第一部分PFOS對(duì)血—腦屏障的損傷效應(yīng)研究 目的：建立PFOS染毒模型，明確PFOS對(duì)腦組織的損傷作用和對(duì)血—腦屏障結(jié)構(gòu)和功能的影響，并分析相關(guān)分子機(jī)制。 方法：一定劑量的PFOS給予ICR雄性小鼠經(jīng)口灌胃染毒，通過體重及臟器系數(shù)觀察一般毒性；運(yùn)用UPLC/MS/MS檢測(cè)血清及腦中的PFOS暴露，分析PFOS通過血—腦屏障的能力；采用光鏡觀察PFOS對(duì)腦組織的損傷作用；采用電鏡觀察PFOS對(duì)血—腦屏障超微結(jié)構(gòu)的破壞；運(yùn)用放免法檢測(cè)血清相關(guān)激素觀察PFOS內(nèi)分泌干擾效應(yīng)；采用免疫印跡和免疫組化檢測(cè)血—腦屏障相關(guān)蛋白的表達(dá)和定位，探討關(guān)鍵的靶分子和相關(guān)分子機(jī)制。 結(jié)果：1.一般毒性：在0~50mg/kg/d劑量下，PFOS未對(duì)動(dòng)物生長(zhǎng)發(fā)育產(chǎn)生顯著影響，腦的臟器系數(shù)也未出現(xiàn)顯著改變。2. PFOS通過血—腦屏障的能力：各PFOS處理組的血清和腦組織樣品中均可檢測(cè)出PFOS；血清PFOS水平與腦中PFOS含量呈顯著正相關(guān)（相關(guān)系數(shù)=0.9676，Pearson，p0.0001）。3.腦組織形態(tài)學(xué)改變：光鏡下，PFOS能夠引起神經(jīng)元細(xì)胞的損傷和星形膠質(zhì)細(xì)胞的水腫；電鏡下，PFOS能夠破壞內(nèi)皮細(xì)胞上的緊密連接，并且引起內(nèi)皮細(xì)胞的內(nèi)質(zhì)網(wǎng)和線粒體的水腫以及神經(jīng)元細(xì)胞的髓鞘水腫。4.血清激素改變：PFOS可顯著誘導(dǎo)動(dòng)物血清四碘甲腺原氨酸（Thyroxine，T4）水平降低，而其它激素如促卵泡生成素（Follicle-stimulating hormone，F(xiàn)SH）、促黃體生成素（Luteinizinghormone，LH）、三碘甲腺原氨酸（Triiodothyronnine，T3）未見顯著改變。5.血—腦屏障相關(guān)蛋白的表達(dá)：①PFOS可下調(diào)腦組織TJ相關(guān)蛋白，各劑量組（0.25、2.5、25和50mg/kg/d）可劑量依賴性地引起ZO-1、 Occludin、Claudin-5和Claudin-11表達(dá)的下降(p0.05或p0.01)；②PFOS可顯著下調(diào)GnRH和GnRHR蛋白的表達(dá)(p0.05或p0.01)，而損傷敏感蛋白S100β的表達(dá)則顯著升高(p0.05或p0.01)，水通道蛋白AQP4的表達(dá)也顯著升高(p0.01)。6.絲裂原活化蛋白激酶（Mitogen-activated protein kinase，MAPK）信號(hào)通路的激活，雖然總的p-38、JNK和Erk的表達(dá)改變不顯著，但磷酸化的p-38、JNK和ERK表達(dá)顯著增加(p0.05或p0.01)。 第二部分PFOS對(duì)星形膠質(zhì)細(xì)胞的影響及機(jī)制研究 目的：探討PFOS對(duì)星形膠質(zhì)細(xì)胞的損傷效應(yīng)，驗(yàn)證MAPK信號(hào)通路激活在其損傷中的作用。 方法：采用細(xì)胞急性毒性實(shí)驗(yàn)摸索染毒劑量；采用免疫印跡法分析相關(guān)蛋白的表達(dá)，驗(yàn)證整體水平的結(jié)果，并探討相關(guān)的分子機(jī)制；運(yùn)用MAPK信號(hào)通路特異性抑制劑聯(lián)合PFOS共同處理星形膠質(zhì)細(xì)胞，采用免疫印跡法分析相關(guān)蛋白表達(dá)的變化，驗(yàn)證MAPK信號(hào)通路相關(guān)蛋白表達(dá)改變與損傷的關(guān)系。 結(jié)果：1.細(xì)胞毒性實(shí)驗(yàn)：在150-200μM的PFOS染毒劑量區(qū)間內(nèi)，，選取不產(chǎn)生明顯毒性的180μM的終濃度進(jìn)行蛋白質(zhì)印跡實(shí)驗(yàn)和MAPK信號(hào)通路特異性抑制劑實(shí)驗(yàn)。2.星形膠質(zhì)細(xì)胞相關(guān)蛋白表達(dá)，PFOS能夠上調(diào)損傷敏感蛋白S100β(p0.01)和水通道蛋白AQP4(p0.05)的表達(dá)。3. MAPK特異性抑制劑與PFOS聯(lián)合處理星形膠質(zhì)細(xì)胞后，p38特異性抑制劑可顯著抑制PFOS誘導(dǎo)的S100β和AQP4蛋白的表達(dá)上調(diào)。 結(jié)論 1. PFOS能夠進(jìn)入腦組織內(nèi)，引起神經(jīng)毒性效應(yīng)并對(duì)BBB造成損傷。 2.緊密連接相關(guān)蛋白ZO-1、Occludin、Claudin-5、Claduin-11以及GnRH、GnRHR、S100β、AQP4可能是PFOS作用的重要的靶分子。 3. p38MAPK信號(hào)通路的激活可能在PFOS誘導(dǎo)的血—腦屏障結(jié)構(gòu)和功能的破壞中起關(guān)鍵作用。PFOS-induced neurotoxicity is still not well known. Therefore, to explore the role ofBBB on PFOS-induced neurotoxicity and the relative molecular mechanisms may behelpful to fully addtress the toxic effects of PFOS on brain. In this study, we investigated the effects and mechanisms of PFOS on BBB invivo and in vitro. The changes of brain morphous, the levels of junction proteinsexpression and locations of junction proteins, as well as the serum levels of hormoneswere estimated. Further more, the level of PFOS in brain tissue and serum were alsodetected. Importantly, the molecular mechanisms of PFOS induced changes of BBBwere deeply explored by a in vitro model. This study will provide an importantreference for environmental hazards assessment, risk assessment and preventionstrategies for humans. Part I: Effects of PFOS on blood-brain barrier in male mice Objective: To reveal the effects of PFOS on BBB and neuroendocrine Methods: The adult male ICR mice were administrated of PFOS by oral for4weeks. Then, the general toxicity including body weight and organ coefficient wereanalyzed. Furthermore, the level of PFOS in brain tissue and serum were detected byUPLC/MS/MS. The morphological changes of brain and/or BBB were analyzed bylight and electron microscopy. The expression and location of proteins related to BBBwere evaluated by immunoblotting and immunohistochemistry analysis. Moreover,detection of the levels of serum hormone were determined by radioimmunoassay. Results:1. General Toxicity: PFOS did not changed the growth and the organcoefficient of brain from0to50mg/kg/d group.2. Serum and brain PFOS levels:PFOS were detected in the serum and brain among the PFOS-treated groups. Thepositive correlation between serum and brain PFOS level was also observed(r=0.9676, Pearson, p0.001).3. Brain Morphology: the significant changes of brainmorphology, damge of neurons and astrocytes edema, were observed in PFOS-treatedgroups. Under electron microscopy, the significant changes including disruption oftight junctions, edema of endoplasmic reticulum, mitochondrial and neuron myelin ofendothelial cells were also observed in PFOS-treated groups.4. Serum Hormones:Compared with the control group, PFOS significantly decreased the levels of serumthyroxine (T4). However, the changes of estradiol(E2), follicle-stimulatinghormone(FSH), luteinizing hormone(LH), triiodothyronnine(T3) were relative slight.5. The expression and localization of proteins related to BBB or neuroendocrinefunction: Compared with control group, PFOS significantly decreased the brain TJproteins expression such as ZO-1, Occludin, Claudin-5and Claudin-11. Similarly,PFOS significantly decreased GnRH and GnRHR in brain (p0.05or p0.01).Furthermore, the expression of damage sensitive protein S100β (p0.05or p0.01)and aquaporin4(p0.01) were significantly increased by PFOS treatment.6.Activation of Mitogen-activated protein kinase (MAPK) pathway: Although theexpression of total p-38, JNK and Erk were not significantly changed by PFOS (p0.05). However, phosphorylation of p-38, JNK and ERK expression was significantlyincreased (p0.05or p0.01). Part II: Molecular mechanisms of astrocytes cells as targetsin PFOS-induced disruption of BBB structure and function Objective: To reveal the role of astrocytes cells on PFOS-induced disruptionof BBB. To assess the relationship between activation of MAPK signaling pathwayand toxic effects of PFOS on BBB. Methods: The toxic effects of PFOS on astrocytes cells were estimated bycytotoxicity analysis. Furthermore, the expression of proteins relatied to BBB inastrocytes cells were detected by analysis. To reveal the role of MAPK signalingpathways in PFOS-induced disruption of BBB, the specific inhibitors of MAPK wereused and the expressions of relative proteins were analyzed. Results:1. Cytotoxicity analysis: The concentration of PFOS range from150to200μM did not exhibit significant toxicity on the astrocytes cells. Therefore,180μM of PFOS was selected and used in the following experiments.2. The expressionof proteins related to the functions of cstrocyte cells: Compared with control group,PFOS significantly increased the expression of damage sensitive protein S100β(p0.01) and aquaporin4(p0.05).3. The role of MAPK signal pathway onPFOS-induced damage of cstrocyte cells: SB203580, a p38specific inhibitor,significantly rescued the increase of S100β and AQP4induced by PFOS. Conclusion 1. PFOS can enter the brain tissue and cause neuroendocrine damage and disruptionof BBB. 2. The protein related to junction and/or neurons and astrocytes damage such as ZO-1,Occludin, Claudin-5, Claudin-11, S100β, GnRH, GnRHR and AQP4might be theimortatnt molecular targets for PFOS in brain tissue. 3. The activation of p38MAPK signaling pathway may be involved in PFOS-induceddisruption of BBB.
[Abstract]:......
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R131

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1 林咸明;譚克平;張愛軍;何麗華;;電針誘導(dǎo)神經(jīng)生長(zhǎng)因子透血腦屏障效應(yīng)及其機(jī)制分析[J];針刺研究;2009年02期

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