自噬相关基因Atg7敲除对三邻甲苯磷酸酯诱导的Neuro-2a细胞轴突损伤的影响
本文选题:三邻甲苯磷酸酯 + 自噬 ; 参考:《山东大学》2017年硕士论文
【摘要】:目的三邻甲苯磷酸酯(Tri-ortho-cresyl phosphate,TOCP)是工业生产中广泛使用的有机磷化合物。人类暴露TOCP可导致一种迟发性神经病(organophosphate-induced delayed neuropathy,OPIDN)的发生,到目前为止其确切发生机制尚不清楚。以前的研究显示OPIDN中神经轴突的病理变化与物理截断引起的轴突wallerian变性相似,即神经轴突自末端向胞体进行性地变性、解体。自噬是真核细胞中负责蛋白质和细胞器清除的主要机制,对于神经元维持自身稳态具有重要作用。众多研究证明神经元自噬异常和轴突变性有关。在本研究中,我们利用自噬相关基因Atg7敲除的自噬缺陷N2a细胞建立中毒模型,研究自噬在TOCP诱导的Neuro-2a(N2a)轴突损伤中的作用,探索TOCP诱导迟发性神经病的发生机制。方法1.细胞毒性实验:CCK8方法测试不同浓度TOCP对N2a细胞增殖的影响,确定OPIDN细胞模型中TOCP染毒剂量。2.细胞分化实验:培养野生型和Atg7基因敲除的自噬缺陷型N2a细胞,分别使用低血清和视黄酸(Retinoic acid,RA)两种方法诱导野生型N2a细胞(Atg7+/+N2a)和自噬缺陷N2a细胞(Atg7-/-N2a),分化24h、48h,直至呈现典型的神经元形态。3.细胞分化能力和轴突损伤情况检测实验:利用0、1.25、2.5、5、10、20μM TOCP处理Atg7+/++N2a细胞,观察毒物对细胞分化能力的影响。在此基础上,以0、5、10μM浓度的TOCP染毒低血清和视黄酸两种方法诱导分化的Atg7+/+N2a细胞和Atg7-/-N2a细胞,观察比较两种N2a细胞轴突变化情况。4.细胞免疫荧光实验:使用免疫荧光法检测0、5、10μM TOCP处理后的Atg7+/+N2a细胞和Atg7-/-N2a细胞中轴突转运蛋白dynaction和自噬标志蛋白LC3B的变化水平。5.蛋白免疫印迹实验:Western blotting检测Atg7+/+N2a细胞和Atg7-/-N2a细胞中 LC3、p62、p-p62、beclin-1、Ub、k48-Ub、k63-Ub 等自噬相关蛋白,kinesin、dynactin等轴浆运输马达蛋白,以及促进轴突损伤相关蛋白SARM1的表达和MAPK信号通路关键分子的磷酸化水平的变化。6.实时荧光 PCR(Quantitative Real-time PCR,Q-PCR)实验:使用 Q-PCR 检测细胞自噬相关基因LC3B、beclin1和转录因子EB的mRNA表达情况,以及轴突变性相关的关键分子 calpain-1、calpain-2、nmnat-2、sarm1、DLK 和 scg10 等基因的mRNA表达水平。结果1.CCK8法检测结果显示,TOCP染毒剂量超过20μM后,随着剂量的增加,对细胞增殖抑制作用越来越明显,直至超过1OO0μM后细胞活性为零。2.低血清和RA两种方法诱导48h之后均可使两种N2a细胞分化成功,即突起长度超过胞体两倍。其中,RA诱导分化的细胞轴突分化长度更明显。3.①Atg7+/+N2a细胞染毒TOCP之后,明显影响了其分化能力,且随着剂量的增加,抑制分化能力越明显。②使用TOCP染毒后,两种细胞轴突长度明显缩短,且随着TOCP染毒剂量增加,轴突损伤逐渐加重。同一剂量下,Atg7-/-N2a细胞轴突缩短程度相较于野生型的小。4.免疫荧光结果显示,随着TOCP染毒剂量的增加,两种细胞中轴突微管马达蛋白Dynactin表达下降,标记自噬泡绿色荧光亮点增多。两种细胞间无显著性差异。5.Western blotting 实验结果显示:Atg7+/+N2a 细胞中 beclin-1 含量减少,LC3-Ⅱ含量明显增加,Atg7-/-N2a细胞中beclin-1同样呈现下降趋势,LC3-Ⅱ不表达。两种细胞中p-p62水平上调,Atg7-/-N2a细胞中p-p62表达量更高。高剂量组两种细胞中轴突运输马达蛋白dynactin和kinesin表达均减少。两种细胞中K48-位点泛素蛋白水平增加,Atg7-/-N2a细胞表达量更高。Atg7+/+N2a细胞中对轴突损伤具有促进性作用的激酶p-p38、p-p42/p44磷酸化水平上升,p-jnk表达下调,蛋白SARM1表达增加,而Atg7-/-N2a细胞中除激酶p-p38呈现上升趋势外,其余均无显著性差异,p-p42/p44在两种细胞间变化具有统计学差异,Atg7+/+N2a细胞磷酸化水平更高。6.荧光定量PCR检测发现:自噬相关基因LC3B、beclin1、转录因子EB的mRNA水平在两种细胞中随着药物剂量的增加而逐步上升。calpain-1在高剂量染毒的Atg7+/+N2a细胞中激活,而在Atg7-/-N2a细胞中calpain-1、calpain-2均被激活。对轴突损伤起保护作用nmnat2基因表达情况是:Atg7+/+N2a细胞逐步上升,而Atg7-/-N2a细胞呈下降趋势,0、5μMTOCP处理组中Atg7-/-N2a细胞表达量更高。同轴突损伤相关的sarm1在高剂量染毒的两种细胞中显著性增加。高剂量组Atg7+/+N2a细胞中DLK含量上升,而Atg7-/-N2a细胞却呈现相反趋势。结论1.TOCP染毒影响了 Atg7+/+N2a和Atg7-/-N2a细胞的分化能力,并能引起已分化的N2a细胞的轴突损伤,其中Atg7+/+N2a细胞的损伤程度比Atg7-/-N2a细胞更加严重。2.TOCP染毒引起N2a细胞自噬激活,而Atg7-/-N2a基因敲除导致细胞自噬活性丧失,同时影响了细胞中促轴突存活和促细胞死亡信号关键因子NMNAT2、Sarm1、DLK 和 MAPK 激酶等。3.自噬基因Atg7敲除能减轻三邻甲苯磷酸酯对Neuro-2a细胞轴突的损伤。
[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.
【学位授予单位】:山东大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R114
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