运动神经元细胞中hSOD1-G93A基因对TBK1的影响

发布时间：2018-04-27 01:10

本文选题：ALS + SOD1　；参考：《河北医科大学》2016年硕士论文

【摘要】：目的:肌萎缩侧索硬化(ALS)是一种致命的、进行性的运动神经元变性病,以运动神经元的功能缺失为主要特征。超氧化物岐化酶1(SOD1)突变假说在ALS发病机制中占有重要地位,尤以突变基因研究最为广泛。本实验以h SOD1-G93A质粒转染运动神经元杂交瘤细胞(NSC34),探索突变SOD1对TANK结合蛋白激酶1(TANK-Binding Kinas,1,TBK1)的影响,为以后的研究打下基础。TBK1是一种广泛表达的蛋白激酶,在细胞的多种生物学功能中发挥着重要作用。TBK1是一种非典型的IΚB激酶(IKK)相关的蛋白激酶。TBK1由729个氨基酸组成,包含4个功能结构域:N末端结构域,泛素样结构域和两个C末端结构域(亮氨酸拉链和螺旋-环-螺旋模体)。TBK1在自噬中起着重要作用,此外也参与固有免疫,清除细菌,抑制细胞生长和增殖。方法:1实验分组本研究中使用稳定转染h SOD1-G3A(突变组)的NSC-34细胞系作为ALS细胞模型,稳定转染空质粒(Empty)、h SOD1-野生型(WT)的NSC-34细胞系作为对照组。2药物干预空质粒组、野生型组和突变组三种细胞系以相似的密度接种于孔板中,分别加入自噬阻断剂巴菲罗霉素A(Bafilomycin A)和蛋白酶体阻断剂MG132。然后分别通过细胞计数试剂盒(cck-8)检测细胞活力;激光共聚焦显微镜观察SOD1突变对p TBK1表达数量的影响;Western Blot检测SOD1、TBK1、p TBK1以及自噬相关蛋白P62、LC3B-II的表达情况。结果:1巴菲罗霉素A和MG132均降低三种细胞的细胞活力,但突变组较空质粒组和野生型组下降明显,差异有统计学意义;2应用激光共聚焦显微镜观察突变组给予自噬阻断剂后较空质粒组p TBK1表达数量降低。3 Western Blot检测发现Bafilomycin A增加自噬相关蛋白P62、LC3B-II的表达水平,此外干预后突变组p TBK1的表达水平较空质粒组降低,差异有统计学意义;空质粒组总的TBK1给予Bafilomycin A后增多(P0.05),但突变组未见明显变化。MG132干预后P62、LC3B-II增多,但突变组p TBK1水平较空质粒组降低,总的TBK1水平没有变化。结论:1突变的SOD1通过自噬和蛋白酶体通路降解。2巴菲罗霉素A干预后,突变组p TBK1较空质粒组降低,说明SOD1突变阻碍TBK1的活化。空质粒组总的TBK1在给予自噬阻断剂后增多,但突变组未见明显变化,推测突变的SOD1阻碍TBK1通过自噬通路降解。3蛋白酶体阻断剂阻断细胞的蛋白酶体通路,间接激活自噬,诱导了TBK1的磷酸化。但是在突变细胞系中,磷酸化TBK1的诱导与空质粒组比较,显著下降,提示了突变SOD1抑制了TBK1的磷酸化。
[Abstract]:Objective: amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease characterized by motor neuron dysfunction. Superoxide dismutase 1 (SOD1) mutation hypothesis plays an important role in the pathogenesis of ALS. In this study, h SOD1-G93A plasmid was transfected into motor neuron hybridoma cell line NSC34A to explore the effect of mutated SOD1 on TANK binding protein kinase (1(TANK-Binding Kinask1). TBK1 was a widely expressed protein kinase. TBK1 is an atypical protein kinase associated with I B kinase. TBK1 consists of 729 amino acids and consists of four functional domains: N-terminal domain. Ubiquitin like domain and two C-terminal domains (leucine zipper and helicyclic helical motif) play an important role in autophagy. In addition, Ubiquitin like domain and two C-terminal domains also participate in innate immunity, remove bacteria, inhibit cell growth and proliferation. Methods in this study, the NSC-34 cell line stably transfected with hSOD1-G3A (mutant group) was used as the ALS cell model, and the NSC-34 cell line stably transfected with empty plasmid (empty plasmid) was used as the control group. Three cell lines of wild type and mutant group were inoculated into the pore plate with similar density. The autophagy blocker A(Bafilomycin A and the proteasome blocker MG132 were added respectively. The effect of SOD1 mutation on the expression of p TBK1 was observed by laser confocal microscopy. The expression of SOD1TBK1C3B-II and autophagy associated protein P62LC3B-II were detected by Western Blot. Results both Bafilomycin A and MG132 decreased the cell viability of the three kinds of cells, but the cell viability of the mutant group was significantly lower than that of the blank plasmid group and the wild type group. The difference was statistically significant using laser confocal microscopy to observe the decrease of p TBK1 expression in mutant group compared with empty plasmid group. The results showed that Bafilomycin A increased the expression level of autophagy associated protein P62L3B-II. In addition, the expression level of p TBK1 in the mutant group was lower than that in the blank plasmid group, and the total TBK1 in the blank plasmid group increased after Bafilomycin A, but no significant change was found in the mutant group. However, the level of p TBK1 in mutant group was lower than that in blank plasmid group, but the total TBK1 level was not changed. Conclusion after the intervention of proteasome pathway and autophagy, the p TBK1 of the mutant group was lower than that of the empty plasmid group, indicating that the SOD1 mutation blocked the activation of TBK1. The total TBK1 of empty plasmid group increased after given autophagy blocker, but there was no obvious change in mutant group. It was speculated that mutant SOD1 blocked TBK1 from blocking proteasome pathway through autophagy pathway and indirectly activated autophagy. The phosphorylation of TBK1 was induced. However, the induction of phosphorylated TBK1 in mutant cell lines was significantly lower than that in blank plasmid group, suggesting that mutant SOD1 inhibited the phosphorylation of TBK1.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R744.8

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