本文選題：三鄰甲苯磷酸酯 + 自噬��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：目的三鄰甲苯磷酸酯(Tri-ortho-cresyl phosphate,TOCP)是工業(yè)生產(chǎn)中廣泛使用的有機(jī)磷化合物。人類暴露TOCP可導(dǎo)致一種遲發(fā)性神經(jīng)病(organophosphate-induced delayed neuropathy,OPIDN)的發(fā)生,到目前為止其確切發(fā)生機(jī)制尚不清楚。以前的研究顯示OPIDN中神經(jīng)軸突的病理變化與物理截?cái)嘁鸬妮S突wallerian變性相似,即神經(jīng)軸突自末端向胞體進(jìn)行性地變性、解體。自噬是真核細(xì)胞中負(fù)責(zé)蛋白質(zhì)和細(xì)胞器清除的主要機(jī)制,對于神經(jīng)元維持自身穩(wěn)態(tài)具有重要作用。眾多研究證明神經(jīng)元自噬異常和軸突變性有關(guān)。在本研究中,我們利用自噬相關(guān)基因Atg7敲除的自噬缺陷N2a細(xì)胞建立中毒模型,研究自噬在TOCP誘導(dǎo)的Neuro-2a(N2a)軸突損傷中的作用,探索TOCP誘導(dǎo)遲發(fā)性神經(jīng)病的發(fā)生機(jī)制。方法1.細(xì)胞毒性實(shí)驗(yàn):CCK8方法測試不同濃度TOCP對N2a細(xì)胞增殖的影響,確定OPIDN細(xì)胞模型中TOCP染毒劑量。2.細(xì)胞分化實(shí)驗(yàn):培養(yǎng)野生型和Atg7基因敲除的自噬缺陷型N2a細(xì)胞,分別使用低血清和視黃酸(Retinoic acid,RA)兩種方法誘導(dǎo)野生型N2a細(xì)胞(Atg7+/+N2a)和自噬缺陷N2a細(xì)胞(Atg7-/-N2a),分化24h、48h,直至呈現(xiàn)典型的神經(jīng)元形態(tài)。3.細(xì)胞分化能力和軸突損傷情況檢測實(shí)驗(yàn):利用0、1.25、2.5、5、10、20μM TOCP處理Atg7+/++N2a細(xì)胞,觀察毒物對細(xì)胞分化能力的影響。在此基礎(chǔ)上,以0、5、10μM濃度的TOCP染毒低血清和視黃酸兩種方法誘導(dǎo)分化的Atg7+/+N2a細(xì)胞和Atg7-/-N2a細(xì)胞,觀察比較兩種N2a細(xì)胞軸突變化情況。4.細(xì)胞免疫熒光實(shí)驗(yàn):使用免疫熒光法檢測0、5、10μM TOCP處理后的Atg7+/+N2a細(xì)胞和Atg7-/-N2a細(xì)胞中軸突轉(zhuǎn)運(yùn)蛋白dynaction和自噬標(biāo)志蛋白LC3B的變化水平。5.蛋白免疫印跡實(shí)驗(yàn):Western blotting檢測Atg7+/+N2a細(xì)胞和Atg7-/-N2a細(xì)胞中 LC3、p62、p-p62、beclin-1、Ub、k48-Ub、k63-Ub 等自噬相關(guān)蛋白,kinesin、dynactin等軸漿運(yùn)輸馬達(dá)蛋白,以及促進(jìn)軸突損傷相關(guān)蛋白SARM1的表達(dá)和MAPK信號(hào)通路關(guān)鍵分子的磷酸化水平的變化。6.實(shí)時(shí)熒光 PCR(Quantitative Real-time PCR,Q-PCR)實(shí)驗(yàn)：使用 Q-PCR 檢測細(xì)胞自噬相關(guān)基因LC3B、beclin1和轉(zhuǎn)錄因子EB的mRNA表達(dá)情況,以及軸突變性相關(guān)的關(guān)鍵分子 calpain-1、calpain-2、nmnat-2、sarm1、DLK 和 scg10 等基因的mRNA表達(dá)水平。結(jié)果1.CCK8法檢測結(jié)果顯示,TOCP染毒劑量超過20μM后,隨著劑量的增加,對細(xì)胞增殖抑制作用越來越明顯,直至超過1OO0μM后細(xì)胞活性為零。2.低血清和RA兩種方法誘導(dǎo)48h之后均可使兩種N2a細(xì)胞分化成功,即突起長度超過胞體兩倍。其中,RA誘導(dǎo)分化的細(xì)胞軸突分化長度更明顯。3.①Atg7+/+N2a細(xì)胞染毒TOCP之后,明顯影響了其分化能力,且隨著劑量的增加,抑制分化能力越明顯。②使用TOCP染毒后,兩種細(xì)胞軸突長度明顯縮短,且隨著TOCP染毒劑量增加,軸突損傷逐漸加重。同一劑量下,Atg7-/-N2a細(xì)胞軸突縮短程度相較于野生型的小。4.免疫熒光結(jié)果顯示,隨著TOCP染毒劑量的增加,兩種細(xì)胞中軸突微管馬達(dá)蛋白Dynactin表達(dá)下降,標(biāo)記自噬泡綠色熒光亮點(diǎn)增多。兩種細(xì)胞間無顯著性差異。5.Western blotting 實(shí)驗(yàn)結(jié)果顯示:Atg7+/+N2a 細(xì)胞中 beclin-1 含量減少,LC3-Ⅱ含量明顯增加,Atg7-/-N2a細(xì)胞中beclin-1同樣呈現(xiàn)下降趨勢,LC3-Ⅱ不表達(dá)。兩種細(xì)胞中p-p62水平上調(diào),Atg7-/-N2a細(xì)胞中p-p62表達(dá)量更高。高劑量組兩種細(xì)胞中軸突運(yùn)輸馬達(dá)蛋白dynactin和kinesin表達(dá)均減少。兩種細(xì)胞中K48-位點(diǎn)泛素蛋白水平增加,Atg7-/-N2a細(xì)胞表達(dá)量更高。Atg7+/+N2a細(xì)胞中對軸突損傷具有促進(jìn)性作用的激酶p-p38、p-p42/p44磷酸化水平上升,p-jnk表達(dá)下調(diào),蛋白SARM1表達(dá)增加,而Atg7-/-N2a細(xì)胞中除激酶p-p38呈現(xiàn)上升趨勢外,其余均無顯著性差異,p-p42/p44在兩種細(xì)胞間變化具有統(tǒng)計(jì)學(xué)差異,Atg7+/+N2a細(xì)胞磷酸化水平更高。6.熒光定量PCR檢測發(fā)現(xiàn):自噬相關(guān)基因LC3B、beclin1、轉(zhuǎn)錄因子EB的mRNA水平在兩種細(xì)胞中隨著藥物劑量的增加而逐步上升。calpain-1在高劑量染毒的Atg7+/+N2a細(xì)胞中激活,而在Atg7-/-N2a細(xì)胞中calpain-1、calpain-2均被激活。對軸突損傷起保護(hù)作用nmnat2基因表達(dá)情況是:Atg7+/+N2a細(xì)胞逐步上升,而Atg7-/-N2a細(xì)胞呈下降趨勢,0、5μMTOCP處理組中Atg7-/-N2a細(xì)胞表達(dá)量更高。同軸突損傷相關(guān)的sarm1在高劑量染毒的兩種細(xì)胞中顯著性增加。高劑量組Atg7+/+N2a細(xì)胞中DLK含量上升,而Atg7-/-N2a細(xì)胞卻呈現(xiàn)相反趨勢。結(jié)論1.TOCP染毒影響了 Atg7+/+N2a和Atg7-/-N2a細(xì)胞的分化能力,并能引起已分化的N2a細(xì)胞的軸突損傷,其中Atg7+/+N2a細(xì)胞的損傷程度比Atg7-/-N2a細(xì)胞更加嚴(yán)重。2.TOCP染毒引起N2a細(xì)胞自噬激活,而Atg7-/-N2a基因敲除導(dǎo)致細(xì)胞自噬活性喪失,同時(shí)影響了細(xì)胞中促軸突存活和促細(xì)胞死亡信號(hào)關(guān)鍵因子NMNAT2、Sarm1、DLK 和 MAPK 激酶等。3.自噬基因Atg7敲除能減輕三鄰甲苯磷酸酯對Neuro-2a細(xì)胞軸突的損傷。
[Abstract]:Objective three Tri-ortho-cresyl phosphate (TOCP) is a widely used organophosphorus compound in industrial production. Human exposure to TOCP can lead to a delayed neuropathy (organophosphate-induced delayed neuropathy, OPIDN). So far, its exact mechanism is not clear. Previous studies showed that OPIDN was in OPIDN. The pathological changes of the axon are similar to the Wallerian degeneration of the axon caused by the physical truncation. That is, the axon is degenerative and disintegrated from the terminal cell body. Autophagy is the main mechanism responsible for the removal of proteins and organelles in eukaryotic cells, which plays an important role in maintaining the homeostasis of the neurons. In this study, we use autophagy related gene Atg7 knockout autophagy deficient N2a cells to establish a poisoned model, study the role of autophagy in TOCP induced Neuro-2a (N2a) axon damage, and explore the mechanism of TOCP induced delayed neuropathy. A square method 1. cytotoxicity test: CCK8 method for testing the different concentrations of TOCP The effect on the proliferation of N2a cells was determined by the determination of.2. cell differentiation at the dose of TOCP in the OPIDN cell model: to cultivate the autophagic deficient N2a cells of the wild type and Atg7 gene knockout, and to induce the wild type N2a cells (Atg7+/+N2a) and the autophagic deficiency N2a cells, respectively, by using the two methods of low serum and retinoic acid (Retinoic acid, RA), respectively. 48h, until a typical neuron morphologic.3. cell differentiation ability and axon damage detection experiment: using 0,1.25,2.5,5,10,20 micron M TOCP to treat Atg7+/++N2a cells and observe the effect of poison on the cell differentiation ability. On this basis, two methods to induce differentiated Atg7+/+N2 with 0,5,10 u M concentration of TOCP low serum and retinoic acid are used to induce the differentiation of Atg7+/+N2. A cells and Atg7-/-N2a cells, observed and compared the changes of the axon of two N2a cells,.4. cell immunofluorescence experiments: the immunofluorescence assay was used to detect the axon transporter dynaction and the autophagy marker protein LC3B of Atg7+/+N2a cells and Atg7-/-N2a cells treated with 0,5,10, M TOCP Ing detected autophagy related proteins such as LC3, p62, p-p62, beclin-1, Ub, k48-Ub, k63-Ub, etc., kinesin, dynactin and other axonal transport motor proteins, as well as the changes in the expression of related proteins and the changes in the phosphorylation level of key molecules in the signaling pathway. -time PCR, Q-PCR) experiment: using Q-PCR to detect the mRNA expression of autophagy related genes LC3B, Beclin1 and transcription factor EB, as well as the expression levels of key molecules related to axon degeneration, calpain-1, calpain-2, nmnat-2, SARM1, EB and other genes. With the increase of dose, the inhibitory effect on cell proliferation is becoming more and more obvious, until the cell activity is zero.2. low serum and RA two methods can induce the two N2a cells to differentiate successfully, that is, the length of the protuberance is more than two times of the cell body. Among them, the differentiation length of the cell axon induced by RA is more obvious.3. (.3.) Atg7+/+N2a cell. After TOCP, the ability of differentiation was obviously affected, and the ability to inhibit differentiation was more obvious with the increase of dose. After the use of TOCP, the axon length of the two kinds of cells shortened obviously, and the axon damage gradually increased with the increase of the dose of TOCP. Under the same dose, the axon shortening of Atg7-/ -N2a cells was smaller than that of the wild type.4.. The results of the immunofluorescence showed that the expression of axon microtubule motor protein Dynactin in the two cells decreased with the increase of the dose of TOCP, and the green fluorescent bright spots of the labeled autophagic vesicles increased. There was no significant difference between the two kinds of cells. The results of.5.Western blotting showed that the content of beclin-1 was decreased in Atg7+/+N2a cells, and the content of LC3- II was significantly increased, Atg7 In -/-N2a cells, beclin-1 also declined, LC3- II was not expressed. The level of p-p62 was up in two cells, and the expression of p-p62 in Atg7-/-N2a cells was higher. The expression of dynactin and kinesin in the axon transport motor protein dynactin and kinesin in the two cells of the high dose group increased. The level of K48- loci was increased in the two cells and the expression of Atg7-/-N2a cells was expressed. In the higher.Atg7+/+N2a cells, the kinase p-p38 that promotes the axon damage, the level of phosphorylation of p-p42/p44 is increased, the expression of p-JNK is down, the expression of protein SARM1 is increased, but there is no significant difference in the rest of the Atg7-/-N2a cells except the kinase p-p38, and p-p42/ p44 is statistically different in the two kinds of cells, Atg7+/+N. The phosphorylation level of 2A cells was higher by.6. fluorescence quantitative PCR detection: the mRNA level of autophagy related genes LC3B, Beclin1, and transcription factor EB increased gradually in the two cells with the increase of drug dosage, and.Calpain-1 was activated in the high dose of Atg7+/+N2a cells, while calpain-1 in Atg7-/ -N2a cells were activated. The expression of nmnat2 gene in the protective effect of sudden injury was that the Atg7+/+N2a cells gradually increased, while the Atg7-/-N2a cells showed a downward trend, and the Atg7-/-N2a cell expression in the 0,5 mu MTOCP treatment group was higher. The SARM1 in the coaxline damage related SARM1 increased significantly in the high dose of the two cells infected. The DLK content in the high dose group Atg7+/+N2a cells increased. But Atg7-/-N2a cells show the opposite trend. Conclusion 1.TOCP affects the differentiation ability of Atg7+/+N2a and Atg7-/-N2a cells and causes the axon damage of the differentiated N2a cells, in which the degree of Atg7+/+N2a cell damage is more serious than that of Atg7-/-N2a cells, which causes the N2a cell autophagy activation by.2.TOCP, and the Atg7-/-N2a gene knocks. The loss of autophagic activity, which also affects the survival of the axons and the key factor of cell death signal NMNAT2, Sarm1, DLK and MAPK kinase, can reduce the damage of three o toluene phosphate to the axon of Neuro-2a cells.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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