本文選題：錳 + 神經(jīng)毒性　； 參考：《第四軍醫(yī)大學》2016年博士論文

【摘要】：背景錳是人體所必須的微量元素。參與人體免疫反應,ATP的生成,骨骼生長等生理反應。此外,錳還可以作為機體許多酶的輔助因子,保障其發(fā)揮正常的生理作用。然而,當機體攝入過多的錳則會引起錳中毒的發(fā)生,其臨床表現(xiàn)主要為類似帕金森氏病病癥。中樞神經(jīng)系統(tǒng)是錳作用人體的主要靶器官,黑質(zhì)紋狀體通路更是錳損傷神經(jīng)系統(tǒng)的關(guān)鍵核團。形成了錳能夠引起黑質(zhì)紋狀體內(nèi)多巴胺能神經(jīng)元功能減弱的經(jīng)典理論。隨著人們對錳神經(jīng)毒性的進一步關(guān)注,錳對海馬腦區(qū)調(diào)控的空間記憶能力損傷也漸漸被人們所證實。研究證實錳在大腦中的蓄積所造成的神經(jīng)毒性作用與阿爾茲海默癥(Alzheimer’s disease,AD)、帕金森癥(Parkinson's disease,PD)、等多種神經(jīng)退行性疾病都有著密切的關(guān)系。在中樞神經(jīng)系統(tǒng)中,小膠質(zhì)細胞是免疫反應的關(guān)鍵細胞,主要來源于腦膜,脈絡叢以及血管周圍。其與巨噬細胞的功能相似,能通過一系列的模式識別受體(Pattern-Recognition Receptors,PRR)實時監(jiān)測中樞神經(jīng)系統(tǒng)的內(nèi)環(huán)境。當組織受損或有害物質(zhì)入侵的情況下,小膠質(zhì)細胞發(fā)生活化反應,一方面發(fā)揮吞噬作用,一方面釋放大量炎癥因子,誘導外周固有免疫細胞及適應性免疫細胞遷移至受損或入侵部位,發(fā)揮免疫防御作用。因此,小膠質(zhì)細胞活化及炎性因子釋放在中樞神經(jīng)系統(tǒng)的免疫應答防御反應中發(fā)揮了重要作用。小膠質(zhì)細胞活化后能夠快速誘導出多種炎性因子,其中包括白介素-1β(interleukin-1beta,il-1β)、腫瘤壞死因子-α(tumornecrosisfactor-alpha,tnf-α)和il-18等。本課題組以往研究發(fā)現(xiàn),錳暴露能夠誘導小膠質(zhì)細胞活化,活化后釋放的炎性因子可能是錳暴露導致神經(jīng)元損傷的關(guān)鍵因素,研究結(jié)果為錳神經(jīng)毒性機制闡明及錳中毒防護提供了重要線索。炎癥體是近年來炎性疾病領(lǐng)域關(guān)注的重點,在炎性因子成熟、釋放過程中發(fā)揮重要作用。nlrp3炎癥體是調(diào)控il-1β、il-18等炎性因子成熟釋放的重要途徑。在神經(jīng)炎癥中,小膠質(zhì)細胞和巨噬細胞內(nèi)的nlrp3炎癥體能被β淀粉樣蛋白(amyloid-beta,aβ),α-共核蛋白(α-synuclein,α-syn)所激活�；罨膎lrp3發(fā)生寡聚化,募集接頭蛋白asc,通多card結(jié)構(gòu)域與pyd結(jié)構(gòu)域相互作用,進一步激活caspase-1,進而加工pro-il-1β為成熟的il-1β,并釋放于細胞外發(fā)揮作用。調(diào)控nlrp3炎癥體活化成熟的因素很多,細胞內(nèi)ros的增多、k+濃度改變、atp水平降低以及自噬等均能夠影響nlrp3炎癥體的活化。自噬是細胞維持內(nèi)環(huán)境穩(wěn)定重要的生理過程。自噬的形成過程包括自噬的啟動、延伸、與溶酶體的融合和降解幾個重要階段。當自噬形成的某一過程受到抑制時則會導致自噬功能處于紊亂狀態(tài)。研究表明,許多神經(jīng)系統(tǒng)疾病的發(fā)生都與自噬功能的異常相關(guān),pd、ad等均發(fā)現(xiàn)體內(nèi)自噬降解過程異常。自噬與nlrp3炎癥體之間的關(guān)系較為復雜,一方面當nlrp3炎癥體處于激活狀態(tài)時,則會促進自噬的發(fā)生;另一方面,自噬的過度活化則能夠抑制nlrp3炎癥體的活化。許多研究表明,自噬的抑制能夠活化nlrp3炎癥體,促進pro-il-1β(36kda)向成熟il-1β轉(zhuǎn)變。雖然自噬能夠調(diào)控nlrp3炎癥體的活化,但其具體機制至今尚未闡明,這也成為當今該領(lǐng)域研究的熱點和難點。目的研究錳暴露誘導的小膠質(zhì)細胞活化及其釋放的炎性因子在錳介導的學習記憶損傷中的作用;揭示nlrp3炎癥體活化是錳誘導小膠質(zhì)細胞活化釋放炎性因子il-1β和il-18的關(guān)鍵環(huán)節(jié);闡明自噬溶酶體功能紊亂與nlrp3炎癥體活化之間的關(guān)系;揭示錳暴露誘導的自噬功能紊亂調(diào)控nlrp3炎癥體活化的具體機制,為錳神經(jīng)毒性的防護和治療提供關(guān)鍵靶點和理論依據(jù)。方法1.通過皮下注射氯化錳的方法,構(gòu)建錳暴露的小鼠體內(nèi)模型,采用原子熒光光譜法檢測小鼠血錳和腦錳的濃度,運用恐懼條件箱以及電生理實驗評估小鼠錳暴露后學習和記憶能力的改變;2.通過免疫熒光化學法檢測錳暴露后nlrp3炎癥體在小鼠海馬區(qū)以及bv2細胞(之后簡稱為bv2)中的表達改變。westernblot檢測nlrp3炎癥體相關(guān)蛋白nlrp3、cleavedcaspase-1,炎性因子il-1β的表達改變;3.通過elisa檢測錳暴露后炎性因子il-1β、il-18和tnf-α的改變,以及qrt-pcr檢測nlrp3和炎性因子il-1β、il-18mrna水平的改變。4.通過免疫熒光化學法檢測自噬相關(guān)蛋白lc3的表達影響,westernblot檢測自噬相關(guān)蛋白beclin1、atg5、lc3、p62、組織蛋白酶b(cathepsinb)蛋白的變化。透射電鏡觀察bv2自噬亞細胞結(jié)構(gòu)的改變;,5.分別運用atg5sirna、bafa1以及nh4cl處理bv2,westernblot檢測錳暴露后nlrp3炎癥體相關(guān)蛋白nlrp3、cleavedcaspase-1,炎性因子il-1β的表達改變,elisa檢測錳暴露后炎性因子il-1β、il-18的改變。結(jié)果1.皮下注射氯化錳7天后,小鼠血錳和腦錳與對照組相比明顯升高,恐懼條件箱檢測以及l(fā)tp實驗發(fā)現(xiàn)錳暴露后能夠引起小鼠學習記憶能力下降;2.海馬腦片以及bv2免疫熒光化學染色表明,錳暴露能夠引起nlrp3炎癥體表達增多。westernblot結(jié)果顯示,體內(nèi)外錳暴露均能夠引起nlrp3炎癥體相關(guān)蛋白nlrp3、cleavedcaspase-1表達的增加;3.elisa實驗檢測發(fā)現(xiàn)錳暴露能夠誘導炎性因子il-1β、il-18表達增加,并且在mrna水平檢測上也得以證實;4.免疫細胞化學熒光染色結(jié)果提示,錳暴露可導致bv2內(nèi)lc3標記的自噬體大量聚集。westernblot發(fā)現(xiàn)與對照組相比,錳暴露后自噬體相關(guān)蛋白beclin1、atg5、lc3ii、p62、組織蛋白酶bbiao’da表達顯著增加。p62升高提示錳暴露能夠引起自噬溶酶體降解功能出現(xiàn)障礙。組織蛋白酶b的表達增加以及電鏡檢測所發(fā)現(xiàn)的溶酶體形態(tài)異常提示錳暴露能夠引起bv2溶酶體功能異常。5.Atg5 siRNA和Baf A1分別抑制自噬體的啟動、延伸和與溶酶體的融合后,并不能抑制錳暴露所誘導的NLRP3炎癥體的活化及IL-1β、IL-18的釋放。NH4Cl作用BV2后,Western blot檢測發(fā)現(xiàn)其能夠降低組織蛋白酶B的表達,抑制錳暴露所誘導的NLRP3炎癥體的活化以及炎性因子IL-1β、IL-18的釋放。結(jié)論1.體內(nèi)實驗證實錳能夠降低海馬腦區(qū)所調(diào)控的學習和記憶能力。2.錳暴露對學習記憶能力的影響可能與其所誘導的小膠質(zhì)細胞活化后釋放的促炎性因子(IL-1β和IL-18等)有關(guān)。3.錳暴露激活NLRP3炎癥體通路是其介導炎性因子釋放的關(guān)鍵因素。4.自噬溶酶體功能紊亂參與了錳誘導的NLRP3炎癥體活化,自噬體啟動、延伸及與溶酶體融合的異常不是調(diào)控NLRP3炎癥體活化的重要環(huán)節(jié).5.錳暴露導致的溶酶體功能異常及cathepsin B的釋放是引起NLRP3炎癥體活化的關(guān)鍵因素。
[Abstract]:Background manganese is a necessary trace element in human body. It participates in human immune response, ATP formation, bone growth and other physiological responses. In addition, manganese can also be used as an auxiliary factor for many enzymes in the body to ensure its normal physiological function. However, excessive manganese intake may lead to the occurrence of manganese poisoning, and its clinical manifestations are mainly similar. Parkinson's disease. The central nervous system is the main target organ of manganese in the human body, and the nigrostriatal pathway is the key nucleus of the manganese damaged nervous system. The classical theory that manganese can cause the dysfunction of dopaminergic neurons in the substantia nigra is a classic theory. With the further attention to the toxicity of manganese, manganese has been used in the hippocampus The neurotoxicity of manganese in the brain has been confirmed. The neurotoxicity of manganese in the brain is closely related to Alzheimer 's disease (AD), Parkinson's disease (PD), and other neurodegenerative diseases. In the central nervous system, small glue is found. The cell is the key cell of the immune response, mainly from the meninges, choroid plexus, and around the blood vessels. It is similar to the function of macrophages and can monitor the internal environment of the central nervous system in real time through a series of pattern recognition receptors (Pattern-Recognition Receptors, PRR). On the one hand, the cell acts as a phagocytosis. On the one hand, it releases a large number of inflammatory factors, and induces the migration of immune cells and adaptive immune cells to the damaged or invasive sites, and plays an immune defense role. Therefore, the activation of microglia and inflammatory factors are released in the immune response defense response of the central nervous system. A variety of inflammatory factors can be induced quickly after the activation of microglia, including -1 beta (interleukin-1beta, IL-1 beta), tumor necrosis factor - alpha (tumornecrosisfactor-alpha, tnf- alpha), and IL-18, etc. this group has previously found that manganese exposure can induce microglia activation and release after activation. Inflammatory factors may be the key factors of neuronal damage caused by manganese exposure. The results provide an important clue to the clarification of manganese neurotoxicity and the protection of manganese poisoning. The inflammatory body is the focus of attention in the field of inflammatory diseases in recent years. The inflammatory factors are mature and play an important role in the release process. The.Nlrp3 inflammatory body is the regulation of IL-1 beta, IL-18 and so on. An important route to mature release of inflammatory factors. In neuropathy, NLRP3 inflammation in microglia and macrophages can be activated by beta amyloid (amyloid-beta, a beta), alpha -synuclein (alpha -synuclein, alpha -syn). Activated NLRP3 oligomerization, recruitment of the head protein ASC, and the interaction of the multi card domain with the PYD domain. One step activates caspase-1, and then processes pro-il-1 beta as the mature IL-1 beta and releases it out of the cell. There are many factors regulating the activation of the NLRP3 inflammatory body, the increase of ROS in the cell, the change of k+ concentration, the decrease of ATP level and autophagy, which can affect the activation of NLRP3 inflammatory body. Autophagy is an important source of the cell maintenance of the internal stability. Process. The process of autophagy consists of the initiation, extension, fusion and degradation of the lysosomes. The autophagy is in disorder when a certain process of autophagy is inhibited. The study shows that the occurrence of many nervous system diseases is associated with autophagic function, PD, ad and so on. The process of autophagic degradation is abnormal. The relationship between autophagy and NLRP3 inflammatory body is more complex. On one hand, when the NLRP3 inflammatory body is activated, autophagy can be promoted; on the other hand, the excessive activation of autophagy can inhibit the activation of the NLRP3 inflammatory body. Many studies have shown that the inhibition of autophagy can activate the NLRP3 inflammatory body and promote the pro- IL-1 beta (36kDa) changes to mature IL-1 beta. Although autophagy can regulate the activation of NLRP3 inflammatory body, its specific mechanism has not yet been elucidated. This has also become a hot and difficult point in this field. The activation of NLRP3 inflammatory body is a key link in the activation and release of inflammatory factors IL-1 beta and IL-18 by manganese induced microglia, and clarifies the relationship between the dysfunction of autophagic lysosome and the activation of NLRP3 inflammatory body, and reveals the body mechanism of the regulation of the dysfunction of autophagy induced by manganese exposure, which regulates the activation of NLRP3 inflammatory bodies, and provides the protection and treatment of manganese neurotoxicity. For the key target and theoretical basis. Method 1. the model of manganese exposed mice was constructed by subcutaneous injection of manganese chloride. The concentration of manganese and manganese in mice was detected by atomic fluorescence spectrometry. The changes of learning and memory ability of mice after manganese exposure were evaluated by using the fear condition box and electrophysiological experiments. 2. by immunofluorescence. The expression changes of NLRP3 inflammatory body in the hippocampus and BV2 cells (later called BV2) after manganese exposure were detected by.Westernblot to detect the changes in the expression of NLRP3 inflammatory proteins NLRP3, cleavedcaspase-1, and inflammatory factor IL-1 beta; 3. the changes of IL-1 beta, IL-18 and tnf- alpha after manganese exposure were detected by ELISA, and the changes in IL-18 and tnf- alpha were detected by ELISA. Detection of NLRP3 and inflammatory factor IL-1 beta, il-18mrna level changes.4. by immunofluorescence chemical assay of autophagy related protein LC3 expression effect, Westernblot detection of autophagy related proteins Beclin1, ATG5, LC3, p62, cathepsin B (CathepsinB) protein changes. Transmission electromicroscope observation of autophagy subcellular structure changes; 5. G5sirna, bafa1 and NH4Cl treated BV2, Westernblot detected the expression of NLRP3 inflammatory body associated protein NLRP3, cleavedcaspase-1, and inflammatory factor IL-1 beta after manganese exposure. ELISA detected the IL-1 beta and IL-18 changes after manganese exposure. Results 1. after 7 days of subcutaneous injection of manganese chloride, the blood manganese and brain manganese in mice were significantly higher than those of the control group. Condition box test and LTP experiment found that manganese exposure could cause the decrease of learning and memory ability in mice. 2. hippocampal slices and BV2 immunofluorescence staining showed that manganese exposure could cause increased expression of NLRP3 inflammatory body.Westernblot, and manganese exposure in vivo and in vivo could lead to NLRP3 inflammation related protein NLRP3, cleavedcaspase-1 3.elisa test found that manganese exposure could induce inflammatory factors IL-1 beta, IL-18 expression increased, and confirmed on mRNA level detection; 4. immunocytochemical fluorescence staining showed that manganese exposure could lead to a large amount of.Westernblot in the autophagic body of LC3 labeled BV2 within BV2 and the manganese exposure was compared with the control group. The expression of phagocytic related proteins Beclin1, ATG5, lc3ii, p62, cathepsin bbiao 'Da expressed a significant increase in.P62, suggesting that manganese exposure can cause the dysfunction of autophagic lysosome degradation. The increase in the expression of cathepsin B and the abnormalities of lysosome morphology found by electron microscopy detection suggest that manganese exposure can cause BV2 lysosome dysfunction.5.At. G5 siRNA and Baf A1 inhibit the activation of autophagosomes, extending and merging with lysosomes, which do not inhibit the activation of NLRP3 inflammatory bodies induced by manganese exposure and IL-1 beta. After the release of.NH4Cl in IL-18, Western blot detection found that the Western blot can reduce the expression of cathepsin B, and inhibit the activation of inflammatory bodies induced by manganese exposure. And the release of inflammatory factors IL-1 beta, IL-18. Conclusion 1. in vivo real manganese can reduce the learning and memory ability of the hippocampus, the effects of.2. manganese exposure on learning and memory ability may be related to the proinflammatory factors (IL-1 beta and IL-18, etc.) released by the induced microglia activation (IL-1 beta and IL-18, etc.), which are related to the activation of NLRP3 inflammation by.3. manganese exposure. The pathway is a key factor in the release of inflammatory factors,.4. autophagic lysosome dysfunction participates in the activation of manganese induced NLRP3 inflammatory bodies, autophagosome initiation, extension, and the abnormality of fusion with lysosomes are not important links to regulate the activation of NLRP3 inflammatory bodies. The dysfunction of the lysosome caused by.5. manganese exposure and the release of cathepsin B is the cause of NLR The key factor in the activation of P3 inflammatory body.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R135.1

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