本文選題：全氟辛酸 + 免疫毒性　； 參考：《杭州師范大學》2015年碩士論文

【摘要】：全氟辛酸(Perfluorooctanoic Acid,PFOA),在世界范圍的水體中均已檢測到,其作為全氟化物(Perfluorinated Compounds,PFCs)的代表物之一,對水生生物造成嚴重威脅,然而,極少有研究關(guān)注PFOA對水生生物免疫系統(tǒng)的影響。本研究選用模式生物斑馬魚(Danio rerio)的免疫器官脾臟和腎臟為研究對象;采用體內(nèi)暴露方法,暴露濃度為0.05,0.1,0.5和1 mg/L,暴露時間為7,14和21天;通過透射電鏡觀察細胞結(jié)構(gòu)變化,定量即時聚合酶鏈鎖反應(yīng)(quantitative Real-time Polymerase Chain Reaction,qRT-PCR)檢測免疫球蛋白,relA轉(zhuǎn)錄因子,髓樣分化因子(Myeloid Differentiation Factor 88,myd88),Toll樣受體2(Toll-Like Receptor 2,TLR2)以及相關(guān)細胞因子mRNA表達水平;此外,利用MATLAB R2013a軟件對試驗數(shù)據(jù)進行三階多項式擬合,利用Excel 2007軟件將免疫球蛋白相對表達量進行雷達三角整合,并分析了各個指標之間相關(guān)性,從而深入研究PFOA致水生生物免疫毒效應(yīng)的潛在致毒機理。透射電鏡結(jié)果顯示:PFOA會對脾臟和腎臟組織結(jié)構(gòu)造成損傷;干擾脾臟免疫細胞脂質(zhì)運輸及代謝,導(dǎo)致其出現(xiàn)脂褐質(zhì)色素顆粒;誘導(dǎo)脾臟和腎臟免疫細胞內(nèi)線粒體及內(nèi)質(zhì)網(wǎng)出現(xiàn)擴張,抑制其進一步增殖、分化及成熟,阻止蛋白質(zhì)合成,甚至導(dǎo)致其凋亡。定量RT-PCR結(jié)果顯示:脾臟和腎臟中的細胞因子和免疫球蛋白的表達量是一種動態(tài)變化過程,它們會隨PFOA暴露劑量和暴露時間的改變而發(fā)生變化;并且這種改變受TLR/NF-κB通路調(diào)控;此外,脾臟中的細胞因子相比腎臟更易受到PFOA干擾。尤其是脾臟中的白介素-1β(Interleukine-1β,IL-1β)表達水平浮動顯著:當0.5 mg/L PFOA暴露14天后,脾臟中IL-1β的相對表達量是對照組的8.1倍;當1 mg/L PFOA暴露21天后,其相對表達量僅是對照組的15%。免疫球蛋白雷達三角圖結(jié)果表明:在低濃度組暴露初期(7天),脾臟相對腎臟,具有較好的穩(wěn)定性,但隨暴露時間延長,暴露劑量增加,脾臟和腎臟的免疫系統(tǒng)均面臨崩潰。線性相關(guān)分析結(jié)果表明:PFOA暴露后,腎臟中的細胞因子主要受TLR/myd88/NF-κB通路調(diào)控,而該通路對脾臟中細胞因子的調(diào)控能力較弱;此外,在脾臟和腎臟中,B細胞活化因子(B Cell Activating Factor,BAFF)是調(diào)控免疫球蛋白的重要細胞因子。MATLAB分析結(jié)果表明:除BAFF以外,PFOA還可通過TLR/myd88/NF-κB通路干擾斑馬魚腎臟中IL-1β、干擾素(Interferon,IFN)、IL-4和IL-21 mRNA的表達;進而影響腎臟中免疫球蛋白表達水平;此外,脾臟中relA的表達水平主要受上游蛋白myd88影響,但NF-κB通路對細胞因子的調(diào)控能力相比腎臟有所不足。綜合上述結(jié)果可得:腎臟中細胞因子和免疫球蛋白的反應(yīng)水平較脾臟具有滯后性,因此可推斷PFOA的首要免疫靶器官是脾臟,并且脾臟中免疫系統(tǒng)的失調(diào)會進一步影響腎臟中免疫系統(tǒng)的平衡,從而最終導(dǎo)致斑馬魚免疫系統(tǒng)整體崩潰。總之,本研究將細胞因子及免疫球蛋白整合考慮,探明PFOA致斑馬魚免疫毒效應(yīng)的潛在致毒機理,為未來防控持久性有機污染物PFOA具有一定的指導(dǎo)意義。
[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.
【學位授予單位】：杭州師范大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：X171.5;X503.225

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