基于斑马鱼模型的全氟辛酸免疫毒效应及机理研究

发布时间：2018-06-03 04:02

本文选题：全氟辛酸 + 免疫毒性　；参考：《杭州师范大学》2015年硕士论文

【摘要】：全氟辛酸(Perfluorooctanoic Acid,PFOA),在世界范围的水体中均已检测到,其作为全氟化物(Perfluorinated Compounds,PFCs)的代表物之一,对水生生物造成严重威胁,然而,极少有研究关注PFOA对水生生物免疫系统的影响。本研究选用模式生物斑马鱼(Danio rerio)的免疫器官脾脏和肾脏为研究对象;采用体内暴露方法,暴露浓度为0.05,0.1,0.5和1 mg/L,暴露时间为7,14和21天;通过透射电镜观察细胞结构变化,定量即时聚合酶链锁反应(quantitative Real-time Polymerase Chain Reaction,qRT-PCR)检测免疫球蛋白,relA转录因子,髓样分化因子(Myeloid Differentiation Factor 88,myd88),Toll样受体2(Toll-Like Receptor 2,TLR2)以及相关细胞因子mRNA表达水平;此外,利用MATLAB R2013a软件对试验数据进行三阶多项式拟合,利用Excel 2007软件将免疫球蛋白相对表达量进行雷达三角整合,并分析了各个指标之间相关性,从而深入研究PFOA致水生生物免疫毒效应的潜在致毒机理。透射电镜结果显示:PFOA会对脾脏和肾脏组织结构造成损伤;干扰脾脏免疫细胞脂质运输及代谢,导致其出现脂褐质色素颗粒;诱导脾脏和肾脏免疫细胞内线粒体及内质网出现扩张,抑制其进一步增殖、分化及成熟,阻止蛋白质合成,甚至导致其凋亡。定量RT-PCR结果显示:脾脏和肾脏中的细胞因子和免疫球蛋白的表达量是一种动态变化过程,它们会随PFOA暴露剂量和暴露时间的改变而发生变化;并且这种改变受TLR/NF-κB通路调控;此外,脾脏中的细胞因子相比肾脏更易受到PFOA干扰。尤其是脾脏中的白介素-1β(Interleukine-1β,IL-1β)表达水平浮动显著:当0.5 mg/L PFOA暴露14天后,脾脏中IL-1β的相对表达量是对照组的8.1倍;当1 mg/L PFOA暴露21天后,其相对表达量仅是对照组的15%。免疫球蛋白雷达三角图结果表明:在低浓度组暴露初期(7天),脾脏相对肾脏,具有较好的稳定性,但随暴露时间延长,暴露剂量增加,脾脏和肾脏的免疫系统均面临崩溃。线性相关分析结果表明:PFOA暴露后,肾脏中的细胞因子主要受TLR/myd88/NF-κB通路调控,而该通路对脾脏中细胞因子的调控能力较弱;此外,在脾脏和肾脏中,B细胞活化因子(B Cell Activating Factor,BAFF)是调控免疫球蛋白的重要细胞因子。MATLAB分析结果表明:除BAFF以外,PFOA还可通过TLR/myd88/NF-κB通路干扰斑马鱼肾脏中IL-1β、干扰素(Interferon,IFN)、IL-4和IL-21 mRNA的表达;进而影响肾脏中免疫球蛋白表达水平;此外,脾脏中relA的表达水平主要受上游蛋白myd88影响,但NF-κB通路对细胞因子的调控能力相比肾脏有所不足。综合上述结果可得:肾脏中细胞因子和免疫球蛋白的反应水平较脾脏具有滞后性,因此可推断PFOA的首要免疫靶器官是脾脏,并且脾脏中免疫系统的失调会进一步影响肾脏中免疫系统的平衡,从而最终导致斑马鱼免疫系统整体崩溃。总之,本研究将细胞因子及免疫球蛋白整合考虑,探明PFOA致斑马鱼免疫毒效应的潜在致毒机理,为未来防控持久性有机污染物PFOA具有一定的指导意义。
[Abstract]:Perfluorooctanoic Acid (PFOA), which has been detected in all water bodies worldwide, is one of the representative of Perfluorinated Compounds (PFCs) and is a serious threat to aquatic organisms. However, few studies have paid attention to the effect of PFOA on the immune system of aquatic organisms. This study selected model biological zebrafish (Dan). The spleen and kidney of immune organs of IO rerio were studied, the exposure concentration was 0.05,0.1,0.5 and 1 mg/L, the exposure time was 7,14 and 21 days, the cell structure changes were observed by transmission electron microscopy, and the quantitative real-time polymerase chain reaction (quantitative Real-time Polymerase Chain Reaction, qRT-PCR) was used to detect the immune globules White, relA transcription factor, myeloid differentiation factor (Myeloid Differentiation Factor 88, MyD88), Toll like receptor 2 (Toll-Like Receptor 2, TLR2) and related cytokine mRNA expression level; in addition, MATLAB R2013a software was used to fit the experimental data by three order polynomial, and the relative expression of immunoglobulin was carried out by 2007 software. The radar triangulation, and analysis of the correlation between the various indexes, has studied the potential toxic mechanism of the immuno effect of PFOA induced aquatic organisms. The transmission electron microscope results show that PFOA can cause damage to the spleen and kidney tissue structure; it interferes with the lipid transport and metabolism of the spleen immune cells, resulting in the appearance of lipofuscin granules; The mitochondria and endoplasmic reticulum in the spleen and kidney immune cells expand, inhibit their further proliferation, differentiation and maturation, prevent protein synthesis, and even lead to their apoptosis. Quantitative RT-PCR results show that the expression of cytokines and immunoglobulin in the spleen and kidney is a dynamic process, they will be exposed to PFOA exposure dose and violence. The change in exposure time changes; and this change is regulated by the TLR/NF- kappa B pathway; in addition, the cytokines in the spleen are more susceptible to PFOA interference than the kidneys. Especially, the level of the expression level of the interleukins -1 beta (Interleukine-1 beta, IL-1 beta) in the spleen is significantly fluctuated: the relative expression of IL-1 beta in the spleen is the relative expression of the spleen when 0.5 mg/L PFOA is exposed. When 1 mg/L PFOA was exposed for 21 days, the relative expression was only the 15%. immunoglobulin radar triangulation of the control group. The results showed that the spleen was relatively stable at the early stage of exposure (7 days) and the spleen was relative to the kidney, but the exposure dose increased and the immune system of the spleen and kidney were in the face of collapse. The results of sex correlation analysis showed that after PFOA exposure, the cytokines in the kidney were mainly regulated by the TLR/myd88/NF- kappa B pathway, and the regulation of the cytokines in the spleen was weak. In addition, in the spleen and kidney, the B cell activating factor (B Cell Activating Factor, BAFF) is an important cytokine.MATLAB analysis of immunoglobulin. The results showed that, except for BAFF, PFOA could also interfere with the expression of IL-1 beta, Interferon, IFN, IL-4 and IL-21 mRNA in the kidney of zebra fish through the TLR/myd88/NF- kappa B pathway, and then affect the expression level of immunoglobulin in the kidney. Besides, the expression level of relA in the spleen is mainly influenced by the upstream protein MyD88. The above results suggest that the reaction level of cytokines and immunoglobulin in the kidney is lagging behind the spleen. Therefore, it can be concluded that the primary immune target organ of PFOA is the spleen, and the imbalance of the immune system in the spleen will further affect the balance of the immune system in the kidney, thus ultimately causing the immune system in the kidney. The overall collapse of zebrafish immune system. In a word, this study considers the integration of cytokine and immunoglobulin, and explores the potential toxic mechanism of PFOA induced zebrafish immune effect, which has a certain guiding significance for the prevention and control of persistent organic pollutants (PFOA) in the future.
【学位授予单位】：杭州师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X171.5;X503.225

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