發(fā)布時(shí)間：2018-04-27 01:10

  本文選題：ALS + SOD1�。� 參考：《河北醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:肌萎縮側(cè)索硬化(ALS)是一種致命的、進(jìn)行性的運(yùn)動(dòng)神經(jīng)元變性病,以運(yùn)動(dòng)神經(jīng)元的功能缺失為主要特征。超氧化物岐化酶1(SOD1)突變假說(shuō)在ALS發(fā)病機(jī)制中占有重要地位,尤以突變基因研究最為廣泛。本實(shí)驗(yàn)以h SOD1-G93A質(zhì)粒轉(zhuǎn)染運(yùn)動(dòng)神經(jīng)元雜交瘤細(xì)胞(NSC34),探索突變SOD1對(duì)TANK結(jié)合蛋白激酶1(TANK-Binding Kinas,1,TBK1)的影響,為以后的研究打下基礎(chǔ)。TBK1是一種廣泛表達(dá)的蛋白激酶,在細(xì)胞的多種生物學(xué)功能中發(fā)揮著重要作用。TBK1是一種非典型的IΚB激酶(IKK)相關(guān)的蛋白激酶。TBK1由729個(gè)氨基酸組成,包含4個(gè)功能結(jié)構(gòu)域:N末端結(jié)構(gòu)域,泛素樣結(jié)構(gòu)域和兩個(gè)C末端結(jié)構(gòu)域(亮氨酸拉鏈和螺旋-環(huán)-螺旋模體)。TBK1在自噬中起著重要作用,此外也參與固有免疫,清除細(xì)菌,抑制細(xì)胞生長(zhǎng)和增殖。方法:1實(shí)驗(yàn)分組本研究中使用穩(wěn)定轉(zhuǎn)染h SOD1-G3A(突變組)的NSC-34細(xì)胞系作為ALS細(xì)胞模型,穩(wěn)定轉(zhuǎn)染空質(zhì)粒(Empty)、h SOD1-野生型(WT)的NSC-34細(xì)胞系作為對(duì)照組。2藥物干預(yù)空質(zhì)粒組、野生型組和突變組三種細(xì)胞系以相似的密度接種于孔板中,分別加入自噬阻斷劑巴菲羅霉素A(Bafilomycin A)和蛋白酶體阻斷劑MG132。然后分別通過(guò)細(xì)胞計(jì)數(shù)試劑盒(cck-8)檢測(cè)細(xì)胞活力;激光共聚焦顯微鏡觀察SOD1突變對(duì)p TBK1表達(dá)數(shù)量的影響;Western Blot檢測(cè)SOD1、TBK1、p TBK1以及自噬相關(guān)蛋白P62、LC3B-II的表達(dá)情況。結(jié)果:1巴菲羅霉素A和MG132均降低三種細(xì)胞的細(xì)胞活力,但突變組較空質(zhì)粒組和野生型組下降明顯,差異有統(tǒng)計(jì)學(xué)意義;2應(yīng)用激光共聚焦顯微鏡觀察突變組給予自噬阻斷劑后較空質(zhì)粒組p TBK1表達(dá)數(shù)量降低。3 Western Blot檢測(cè)發(fā)現(xiàn)Bafilomycin A增加自噬相關(guān)蛋白P62、LC3B-II的表達(dá)水平,此外干預(yù)后突變組p TBK1的表達(dá)水平較空質(zhì)粒組降低,差異有統(tǒng)計(jì)學(xué)意義;空質(zhì)粒組總的TBK1給予Bafilomycin A后增多(P0.05),但突變組未見明顯變化。MG132干預(yù)后P62、LC3B-II增多,但突變組p TBK1水平較空質(zhì)粒組降低,總的TBK1水平?jīng)]有變化。結(jié)論:1突變的SOD1通過(guò)自噬和蛋白酶體通路降解。2巴菲羅霉素A干預(yù)后,突變組p TBK1較空質(zhì)粒組降低,說(shuō)明SOD1突變阻礙TBK1的活化。空質(zhì)粒組總的TBK1在給予自噬阻斷劑后增多,但突變組未見明顯變化,推測(cè)突變的SOD1阻礙TBK1通過(guò)自噬通路降解。3蛋白酶體阻斷劑阻斷細(xì)胞的蛋白酶體通路,間接激活自噬,誘導(dǎo)了TBK1的磷酸化。但是在突變細(xì)胞系中,磷酸化TBK1的誘導(dǎo)與空質(zhì)粒組比較,顯著下降,提示了突變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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R744.8

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本文編號(hào)：1808495