PFOS对小鼠血—脑屏障的损伤效应及机制研究

发布时间：2018-02-14 14:07

本文关键词： 全氟辛烷磺酸血—脑屏障 MAPK信号通路 ICR小鼠星形胶质细胞　出处：《南京医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：全氟辛烷磺酸/盐（Perfluorooctane sulfonate，PFOS）是一类近年来引起广泛关注的新型环境持久性有机污染物（Persist organic pollutants，POPs）。PFOS类化合物具有难降解性、生物蓄积性和食物链放大作用等，其所造成的环境污染已遍及全球生态系统，成为近年来学界的研究热点。PFOS不但可诱导肝脏、免疫及内分泌系统的损害，而且对动物的神经发育也有影响。近年来在PFOS神经毒性研究中，主要关注了PFOS对各类神经元细胞的作用以及经此途径诱导神经损伤的相关分子机制。血—脑屏障是存在于脑和脊髓内的毛细血管与神经组织之间的一个调节中枢神经系统内环境的细胞屏障，能够维持脑内离子、激素和递质等的动态平衡。血—脑屏障能够被多种外源性因素所影响。目前研究较多的是重金属、电离辐射和创伤等，外源性化学物对血—脑屏障的影响研究较少。PFOS若能够对神经元细胞造成损伤，血—脑屏障可能是关键环节，因此，阐明PFOS对血—脑屏障的影响及其分子机制有助于全面认识PFOS诱导的神经损伤过程。为此，我们利用ICR雄性小鼠作为PFOS的染毒模型，通过检测脑中PFOS含量、观察脑组织的形态学改变情况、紧密连接相关蛋白和损伤相关蛋白的表达情况以及血清中相关激素水平等，系统评估PFOS对血—脑屏障的结构和功能的影响，寻找PFOS所致损伤效应过程中的靶细胞和靶分子，并通过体外研究进一步探讨PFOS影响血—脑屏障的分子机制。为环境持久性有机污染物的危害评价、风险评估以及预防和控制策略制定提供重要参考。第一部分PFOS对血—脑屏障的损伤效应研究目的：建立PFOS染毒模型，明确PFOS对脑组织的损伤作用和对血—脑屏障结构和功能的影响，并分析相关分子机制。方法：一定剂量的PFOS给予ICR雄性小鼠经口灌胃染毒，通过体重及脏器系数观察一般毒性；运用UPLC/MS/MS检测血清及脑中的PFOS暴露，分析PFOS通过血—脑屏障的能力；采用光镜观察PFOS对脑组织的损伤作用；采用电镜观察PFOS对血—脑屏障超微结构的破坏；运用放免法检测血清相关激素观察PFOS内分泌干扰效应；采用免疫印迹和免疫组化检测血—脑屏障相关蛋白的表达和定位，探讨关键的靶分子和相关分子机制。结果：1.一般毒性：在0~50mg/kg/d剂量下，PFOS未对动物生长发育产生显著影响，脑的脏器系数也未出现显著改变。2. PFOS通过血—脑屏障的能力：各PFOS处理组的血清和脑组织样品中均可检测出PFOS；血清PFOS水平与脑中PFOS含量呈显著正相关（相关系数=0.9676，Pearson，p0.0001）。3.脑组织形态学改变：光镜下，PFOS能够引起神经元细胞的损伤和星形胶质细胞的水肿；电镜下，PFOS能够破坏内皮细胞上的紧密连接，并且引起内皮细胞的内质网和线粒体的水肿以及神经元细胞的髓鞘水肿。4.血清激素改变：PFOS可显著诱导动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，FSH）、促黄体生成素（Luteinizinghormone，LH）、三碘甲腺原氨酸（Triiodothyronnine，T3）未见显著改变。5.血—脑屏障相关蛋白的表达：①PFOS可下调脑组织TJ相关蛋白，各剂量组（0.25、2.5、25和50mg/kg/d）可剂量依赖性地引起ZO-1、 Occludin、Claudin-5和Claudin-11表达的下降(p0.05或p0.01)；②PFOS可显著下调GnRH和GnRHR蛋白的表达(p0.05或p0.01)，而损伤敏感蛋白S100β的表达则显著升高(p0.05或p0.01)，水通道蛋白AQP4的表达也显著升高(p0.01)。6.丝裂原活化蛋白激酶（Mitogen-activated protein kinase，MAPK）信号通路的激活，虽然总的p-38、JNK和Erk的表达改变不显著，但磷酸化的p-38、JNK和ERK表达显著增加(p0.05或p0.01)。第二部分PFOS对星形胶质细胞的影响及机制研究目的：探讨PFOS对星形胶质细胞的损伤效应，验证MAPK信号通路激活在其损伤中的作用。方法：采用细胞急性毒性实验摸索染毒剂量；采用免疫印迹法分析相关蛋白的表达，验证整体水平的结果，并探讨相关的分子机制；运用MAPK信号通路特异性抑制剂联合PFOS共同处理星形胶质细胞，采用免疫印迹法分析相关蛋白表达的变化，验证MAPK信号通路相关蛋白表达改变与损伤的关系。结果：1.细胞毒性实验：在150-200μM的PFOS染毒剂量区间内，，选取不产生明显毒性的180μM的终浓度进行蛋白质印迹实验和MAPK信号通路特异性抑制剂实验。2.星形胶质细胞相关蛋白表达，PFOS能够上调损伤敏感蛋白S100β(p0.01)和水通道蛋白AQP4(p0.05)的表达。3. MAPK特异性抑制剂与PFOS联合处理星形胶质细胞后，p38特异性抑制剂可显著抑制PFOS诱导的S100β和AQP4蛋白的表达上调。结论 1. PFOS能够进入脑组织内，引起神经毒性效应并对BBB造成损伤。 2.紧密连接相关蛋白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信号通路的激活可能在PFOS诱导的血—脑屏障结构和功能的破坏中起关键作用。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]:......
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R131

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