本文選題：應(yīng)激顆粒 + 丙二酸鈉��； 參考：《天津醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的:生物體生活在一個(gè)復(fù)雜的、多變的環(huán)境中,外界環(huán)境中也存在許多有害刺激,如:熱休克、冷休克、氧化應(yīng)激、病毒感染和UV照射等,真核細(xì)胞為了適應(yīng)這些外界刺激,進(jìn)化出了一系列復(fù)雜的應(yīng)激系統(tǒng),包括:應(yīng)激顆粒(Stress Granules,SGs),加工體(Processing Bodies,PBs),線粒體應(yīng)激,內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic reticulum Stress,ERS)等。然而在應(yīng)激情況下去探討細(xì)胞防御機(jī)制之間的功能聯(lián)系是非常有意義的,有研究表明應(yīng)激顆粒與內(nèi)質(zhì)網(wǎng)應(yīng)激、線粒體應(yīng)激之間有一定的聯(lián)系。同時(shí)也有研究證明,在一種內(nèi)質(zhì)網(wǎng)鈣泵的抑制劑毒胡蘿卜素的刺激下,TAR DNA結(jié)合蛋白43(TAR DNA binding protein 43,TDP-43)和staufen 1蛋白被招募到應(yīng)激顆粒中,而內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)應(yīng)激顆粒形成的機(jī)制已有文獻(xiàn)報(bào)導(dǎo)。一些線粒體呼吸鏈抑制劑或解偶聯(lián)劑等可以引起線粒體應(yīng)激,同時(shí)有研究證明FCCP與百枯草還能誘導(dǎo)應(yīng)激顆粒的形成,然而線粒體應(yīng)激與應(yīng)激顆粒形成之間的相關(guān)性,卻沒有明確的文獻(xiàn)報(bào)導(dǎo),因此本課題旨在探討線粒體應(yīng)激與應(yīng)激顆粒形成之間的分子機(jī)制。方法:本研究分為三大部分,第一部分:(1)通過細(xì)胞增殖實(shí)驗(yàn)和細(xì)胞免疫熒光(Immunofluorescence,IF)確定丙二酸鈉引起SGs形成的最適濃度;(2)通過細(xì)胞免疫熒光分析SGs標(biāo)志性蛋白的共定位情況;(3)通過檢測(cè)線粒體三磷酸腺苷(adenosine triphosphate,ATP)、活性氧(Reactive oxygen species,ROS)、膜電位(mitochonrial membrane potential,MMP)以及形態(tài)的改變,證明丙二酸鈉對(duì)線粒體功能的影響;(4)利用細(xì)胞免疫熒光、活細(xì)胞工作系統(tǒng)等技術(shù),綜合分析SGs與線粒體的定位現(xiàn)象。第二部分:(1)利用多聚核糖體蔗糖密度梯度離心分析SGs形成與翻譯抑制的相關(guān)性;(2)通過蛋白質(zhì)印跡法,小干擾RNA(small interfering RNA,siRNA)轉(zhuǎn)染,細(xì)胞免疫熒光等綜合分析真核翻譯起始因子2α(eukaryotic Initiation Factor2α,eIF2α)與SGs形成的相關(guān)性;(3)通過蛋白印跡,結(jié)合si RNA干擾、細(xì)胞免疫熒光分析真核起始因子4E結(jié)合蛋白(eIF4E binding protein,4EBP1)與SGs形成的相關(guān)性;(4)通過蛋白印跡,結(jié)合激酶抑制劑、細(xì)胞免疫熒光分析絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)途徑與SGs形成間的相關(guān)性。第三部分:(1)通過Cell Titer-Glo?螢光細(xì)胞活性,蛋白印跡,免疫熒光,凋亡及細(xì)胞增殖的檢測(cè)來探討ATP與SGs形成之間的關(guān)系;(2)通過ROS檢測(cè),蛋白印跡,免疫熒光,細(xì)胞凋亡及增殖的檢測(cè)來探討ROS與SGs形成之間的關(guān)系。結(jié)果:第一部分:(1)當(dāng)丙二酸鈉的刺激濃度為0~200 mM時(shí),細(xì)胞的增殖活性并未受到明顯影響。在丙二酸鈉濃度為50 mM時(shí),胞漿內(nèi)開始出現(xiàn)細(xì)小的SGs,當(dāng)藥物濃度增大到100 mM時(shí),出現(xiàn)的SGs最為明顯,濃度繼續(xù)增大后SGs逐漸消失。(2)濃度為100 mM的丙二酸鈉刺激后,HeLa細(xì)胞內(nèi)源性的SGs標(biāo)志性蛋白R(shí)as-GTP酶激活蛋白SH3結(jié)構(gòu)域結(jié)合蛋白(Ras-GTPase-activating protein SH3domain-binding protein,G3BP)分別和T細(xì)胞胞內(nèi)抗原相關(guān)蛋白(TIA-1-related protein,TIAR)、T細(xì)胞胞內(nèi)抗原1(T cell intracellular antigen 1,TIA-1)、人類抗原R(human antigen R,HuR)蛋白有共定位的現(xiàn)象。(3)與未處理組相比,100 mM丙二酸鈉導(dǎo)致活性氧增多、膜電位降低、ATP減少。在未刺激時(shí),線粒體呈現(xiàn)長(zhǎng)條網(wǎng)絡(luò)狀結(jié)構(gòu),刺激后皺縮成小的團(tuán)塊。(4)在丙二酸鈉刺激后,線粒體聚縮成為團(tuán)塊狀,并在胞核周圍聚集,形成的SGs聚集在線粒體的周圍。第二部分:(1)與對(duì)照組相比,亞砷酸鈉與丙二酸鈉刺激時(shí),多聚核糖體都出現(xiàn)了解聚的現(xiàn)象。(2)在丙二酸鈉處理后,顯著提高了eIF2α蛋白的磷酸化水平。利用RNA干擾技術(shù),下調(diào)細(xì)胞內(nèi)源性的蛋白激酶GCN2(general control nonderepressible 2,GCN2)或血紅素調(diào)節(jié)的起始因子2α(heme-regulated initiation factor 2αkinase,HRI)的含量后,降低了eIF2α的磷酸化水平,蛋白激酶R(protein kinase R,PKR)和類蛋白激酶R的內(nèi)質(zhì)網(wǎng)激酶(PKR-like endoplasmic reticulum kinase,PERK)對(duì)其影響不大,但應(yīng)激時(shí)GCN2或HRI這兩個(gè)蛋白的敲低并未影響SGs的形成。(3)應(yīng)激狀態(tài)下,4EBP1的磷酸化被抑制,當(dāng)下調(diào)細(xì)胞內(nèi)4EBP1蛋白表達(dá)時(shí),形成的SGs顆粒變少,體積減小。(4)應(yīng)激時(shí),MAPK途徑的p38、細(xì)胞外調(diào)節(jié)蛋白激酶1/2(extracellular regulated protein kinases,Erk1/2)和c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)都被激活,當(dāng)分別使用p38、Erk1/2、JNK蛋白的抑制劑SB203580、PD98059、SP600125處理后,SGs的形成并未發(fā)現(xiàn)明顯改變。第三部分:(1)在丙二酸鈉刺激情況下,提高細(xì)胞內(nèi)ATP的含量時(shí),形成SGs的數(shù)目不變,但體積有所增大。丙二酸鈉處理后,補(bǔ)充或者不補(bǔ)充ATP都能引起4EBP1磷酸化的降低和eIF2α磷酸化的增強(qiáng),然而ATP的補(bǔ)充與否并未引起eIF2α和4EBP1磷酸化狀態(tài)的改變。在應(yīng)激后,切割的caspase 3增多,凋亡增多,然而ATP的補(bǔ)充并未影響切割的caspase 3的含量以及凋亡情況,也未影響其細(xì)胞的增殖情況。(2)在丙二酸鈉刺激情況下,當(dāng)使用N-乙酰半胱氨酸(N-Acetyl Cysteine,NAC)降低ROS的水平后,形成SGs的數(shù)目、體積均未發(fā)生顯著改變,與此同時(shí)eIF2α和4EBP1的磷酸化狀態(tài)也沒有明顯變化。在應(yīng)激后,ROS的清除并未影響切割的caspase 3的含量和凋亡情況,細(xì)胞的增殖情況也未受到影響。結(jié)論:1)在丙二酸鈉刺激情況下,SGs標(biāo)志性蛋白G3BP蛋白和TIAR、TIA-1、HuR三種SGs相關(guān)蛋白完全共定位,細(xì)胞胞漿中形成SGs。同時(shí)線粒體的相關(guān)功能指標(biāo),如:ATP、ROS、MMP、形態(tài)等都發(fā)生了明顯改變,證明線粒體發(fā)生了功能紊亂。2)應(yīng)激時(shí),發(fā)生eIF2α、4EBP1和MAPK磷酸化的變化,其中4EBP1磷酸化的改變是誘導(dǎo)SGs形成的關(guān)鍵因素,eIF2α與MAPK的磷酸化并不是起主導(dǎo)作用。3)在刺激時(shí),ATP的補(bǔ)充能促進(jìn)SGs體積增大,此改變并不是通過eIF2α、4EBP1起作用的,ATP水平的改變對(duì)細(xì)胞的增殖和凋亡沒有顯著影響。而清除ROS后對(duì)SGs的體積和數(shù)目,細(xì)胞的增殖和凋亡也沒有明顯影響。
[Abstract]:Objective: living organisms live in a complex and changeable environment, and there are many harmful stimuli in the external environment, such as heat shock, cold shock, oxidative stress, virus infection and UV irradiation. In order to adapt to these external stimuli, eukaryotic cells have evolved a series of complex stress systems, including stress particles (Stress Granules, SGs). Processing Bodies (PBs), mitochondrial stress, endoplasmic reticulum stress (Endoplasmic reticulum Stress, ERS) and so on. However, it is very meaningful to explore the functional connections between cell defense mechanisms under stress. Some studies have shown that stress particles are excited by endoplasmic reticulum, and there is a certain relationship between mitochondrial stress. The TAR DNA binding protein 43 (TAR DNA binding protein 43, TDP-43) and Staufen 1 protein were recruited to stress particles under the stimulation of a kind of endoplasmic reticulum calcium pump inhibitor. The mechanism of endoplasmic reticulum stress induced stress particles formation has been reported in the literature. Some mitochondrial respiratory chain inhibitors or uncoupling agents are available. In order to induce mitochondrial stress, some studies have shown that FCCP and 100 withered grass can also induce the formation of stress particles. However, there is no clear literature on the correlation between mitochondrial stress and the formation of stress particles. Therefore, this study aims to explore the molecular mechanism between mitochondrial stress and stress particles formation. Methods: This study is divided into three Most, the first part: (1) the optimum concentration of SGs formation caused by sodium malonate was determined by cell proliferation experiment and cell immunofluorescence (Immunofluorescence, IF); (2) the co localization of SGs markers by cell immunofluorescence; (3) through the detection of mitochondrial ATP (adenosine triphosphate, ATP), reactive oxygen species (Reactive). Oxygen species, ROS), membrane potential (mitochonrial membrane potential, MMP) and morphological changes, which prove the effect of sodium malonate on mitochondrial function; (4) using cell immunofluorescence, living cell working system and other techniques to analyze the localization of SGs and mitochondria synthetically. The second part: (1) use polyribosome sucrose density gradient centrifugation The correlation between SGs formation and translation inhibition was analyzed. (2) the correlation between eukaryotic translation initiation factor 2 alpha (eukaryotic Initiation Factor2 a, eIF2 a) and SGs formation was analyzed by Western blot, transfection of small interference RNA (small interfering RNA, siRNA), cell immunofluorescence and so on. (3) immunoblotting, combined with Si interference, cell immunity The correlation between the fluorescence analysis of the eukaryotic initiation factor 4E binding protein (eIF4E binding protein, 4EBP1) and the formation of SGs; (4) the correlation between mitogen activated protein kinase (mitogen-activated protein kinase, MAPK) pathway and SGs formation through Western blot and kinase inhibitor, cell immunofluorescence analysis (mitogen-activated protein kinase, MAPK). The third part: (1) through Cell Glo? Fluorescence cell activity, Western blot, immunofluorescence, apoptosis and cell proliferation detection to explore the relationship between ATP and SGs formation; (2) the relationship between ROS and SGs formation through detection of ROS, Western blot, immunofluorescence, apoptosis and proliferation. Results: (1) the stimulation concentration of sodium malonate is 0~200 mM When the concentration of sodium malonate was 50 mM, a small SGs began to appear in the cytoplasm. When the concentration of the drug increased to 100 mM, the SGs was most obvious. The concentration continued to increase and SGs gradually disappeared. (2) the endogenous SGs marker protein Ras in HeLa cells was stimulated by the concentration of 100 mM. The -GTP protein SH3domain-binding protein (G3BP) and the intracellular antigen related proteins (TIA-1-related protein, TIAR) of the T cells (TIA-1-related protein, TIAR), and the protein of the intracellular antigen 1 of the T cells (3). Compared with the untreated group, 100 mM sodium malonate leads to the increase of reactive oxygen species, the decrease of membrane potential and the decrease of ATP. In the absence of stimulation, the mitochondria present a long strip network structure and then shrink into small lumps. (4) after the stimulation of sodium malonate, the mitochondria are condensed into mass, and the formation of SGs is gathered around the mitochondria. The two part: (1) compared with the control group, when sodium arsenite and sodium malonate were stimulated by sodium arsenite and sodium malonate, the accumulation of polyribosomes all appeared. (2) after the treatment of sodium malonate, the phosphorylation level of eIF2 alpha protein was significantly improved. RNA interference technique was used to reduce the endogenous egg white kinase GCN2 (general control nonderepressible 2, GCN2) or blood red. After the content of the starting factor 2 alpha (heme-regulated initiation factor 2 alpha kinase, HRI), the level of phosphorylation of eIF2 alpha is reduced, and the protein kinase R (protein kinase R, PKR) and the protein kinase R like endoplasmic reticulum kinase have little effect on it, but the two proteins are stressed during stress. The knock down did not affect the formation of SGs. (3) the phosphorylation of 4EBP1 was inhibited under stress. When the expression of 4EBP1 protein was downregulated, the formation of SGs particles became less and the volume decreased. (4) when stress was stressed, the p38 of the MAPK pathway, the extracellular regulated protein kinases, Erk1/2) and the terminal amino terminal kinase Rminal kinase, JNK) were activated, and when the inhibitors SB203580, PD98059, and SP600125 were treated with p38, Erk1/2, and JNK protein, the formation of SGs was not obviously changed. Third: (1) when the sodium propanate was stimulated, the number of SGs was increased, but the volume increased. After sodium malonate treatment, Supplementation or without supplementation of ATP could cause the decrease of phosphorylation of 4EBP1 and the enhancement of eIF2 alpha phosphorylation, however, the supplementation of ATP did not cause changes in the state of phosphorylation of eIF2 A and 4EBP1. After stress, the number of caspase 3 increased and apoptosis increased. However, ATP supplementation did not affect the content of caspase 3 and apoptosis, and no shadow of ATP. (2) under the stimulation of sodium malonate, when N- acetylcysteine (N-Acetyl Cysteine, NAC) was used to reduce the level of ROS, the number of SGs was formed, and the volume of SGs did not change significantly. At the same time, there was no significant change in the phosphorylation of eIF2 A and 4EBP1. After stress, the clearance of ROS did not affect the cutting C. The content and apoptosis of Aspase 3 and the cell proliferation were also not affected. Conclusion: 1) under the condition of sodium malonate, the SGs marker protein G3BP protein and the three SGs related proteins of TIAR, TIA-1, HuR are completely Co located, and the cellular cytoplasm forms SGs. and the mitochondrial function indexes, such as ATP, ROS, MMP, morphology, etc. The change of eIF2 alpha, 4EBP1 and MAPK phosphorylation, in which the phosphorylation of 4EBP1 is the key factor in the induction of SGs formation, the phosphorylation of 4EBP1 is not the dominant function of.3) the phosphorylation of eIF2 A and MAPK does not play a dominant role in the stimulation of the 4EBP1. The ATP supplement can increase the SGs volume, and this change does not pass through eIF2 alpha. The effects of 4EBP1 on the proliferation and apoptosis of cells were not significantly affected by the changes of ATP level, but the clearance of ROS had no significant effect on the volume and number of SGs, the proliferation and apoptosis of the cells.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R329.2

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