本文選題：GA6　切入點(diǎn)：自噬　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:自噬是從真核生物到人都高度保守的一種細(xì)胞自我消耗的途徑。在饑餓條件下,細(xì)胞通過(guò)自噬途徑降解自身蛋白和細(xì)胞器得到核苷酸、氨基酸、脂肪酸、糖類(lèi)和ATP,以維持細(xì)胞代謝的正常進(jìn)行而得以在惡劣環(huán)境下繼續(xù)存活,自噬還能清除細(xì)胞內(nèi)錯(cuò)誤折疊的蛋白和破損的細(xì)胞器,維持胞內(nèi)蛋白和細(xì)胞器處于正常水平。自噬的適時(shí)發(fā)生和適當(dāng)?shù)淖允伤绞菣C(jī)體正常運(yùn)作的一大保證。自噬異常與炎癥、腫瘤和神經(jīng)退行性疾病等多種疾病有關(guān),目前靶向自噬的藥物3-甲基腺嘌呤和氯喹都已經(jīng)被用于腫瘤的治療,對(duì)自噬的深入了解能夠輔助醫(yī)療,指導(dǎo)藥物的開(kāi)發(fā)和臨床用藥,促進(jìn)新的治療策略的產(chǎn)生。自噬流的主要檢測(cè)指標(biāo)包括pro-LC3向LC3-I的轉(zhuǎn)化、LC3-I向LC3-II的轉(zhuǎn)化,P62在自噬溶酶體中的降解。自噬促進(jìn)時(shí),pro-LC3的切割增加,LC3-I的脂化增加,P62的降解增加。自噬事件包括自噬的起始、成核、自噬前體膜的延長(zhǎng)、自噬體的成熟、自噬體與溶酶體的融合、吞噬物的降解再利用。自噬前體膜的延長(zhǎng)涉及到pro-LC3在半胱氨酸酶Atg4B的作用下被切割掉C末端的5個(gè)氨基酸(MKLSV)變成LC3-I,LC3-I經(jīng)Atg7、Atg3、Atg10、Atg12-Atg5-Atg16L的共同作用被脂化形成磷脂酰乙醇胺(PE,Phosphatidylethanolamine)修飾形式的LC3-II,并特異性定位于自噬前體膜上,這為自噬體的成熟創(chuàng)造條件。但這一切割和脂化過(guò)程被如何抑制還不清楚,因此本研究擬探討本實(shí)驗(yàn)室發(fā)現(xiàn)的GA6癌蛋白在這一切割和脂化過(guò)程中的作用及GA6對(duì)自噬的影響。內(nèi)容:本研究的研究?jī)?nèi)容主要包括如下:1)探討GA6是否與自噬有關(guān)。篩選與GA6有相互作用的蛋白,并進(jìn)行驗(yàn)證。2)GA6對(duì)自噬有什么影響。GA6是促進(jìn)自噬還是抑制自噬,有哪些表現(xiàn)可以證明。3)GA6對(duì)LC3切割的影響。GA6對(duì)LC3的切割有什么影響,具體影響機(jī)制是什么。4)GA6對(duì)LC3-I脂化的影響。GA6對(duì)LC3-I的脂化有什么影響,該影響是否因?yàn)長(zhǎng)C3-I的形成變化引起。5)GA6對(duì)自噬的影響在C57小鼠上有什么表型。結(jié)合(60)Co照射,分析GA6、自噬與放射敏感性的關(guān)系。方法:利用CRISPR/Cas9技術(shù)構(gòu)建了GA6-/-的乳腺癌細(xì)胞亞株ZR75-1和GA6-/-C57小鼠,并確定了能夠有效敲低GA6的siRNA,建立了敲低GA6的宮頸癌細(xì)胞亞株Hela。利用免疫共沉淀結(jié)合質(zhì)譜法和快速蛋白液相色譜法篩選與GA6有關(guān)的自噬蛋白,并與GA6進(jìn)行Co-IP,進(jìn)一步判斷其是否與GA6具有相互作用,接著我們用GST pulldown實(shí)驗(yàn)最終確定具體是哪個(gè)自噬蛋白與GA6有直接相互作用,并確定此自噬蛋白為本研究的中心。應(yīng)用免疫熒光技術(shù)觀察點(diǎn)狀LC3的形成情況,應(yīng)用透射電子顯微鏡技術(shù)觀察自噬體和自噬溶酶體的形成情況,應(yīng)用Western blot技術(shù)檢測(cè)LC3-I向LC3-II的轉(zhuǎn)化情況。通過(guò)氯喹和雷帕霉素的處理,探究GA6對(duì)自噬的影響具體表現(xiàn)在哪個(gè)自噬環(huán)節(jié),又是否通過(guò)mTOR通路。為探討GA6對(duì)LC3的切割和脂化的影響,我們構(gòu)建了Myc-LC3-PLA2的重組質(zhì)粒,在體內(nèi)研究GA6是否影響LC3的切割;構(gòu)建了GST-LC3-GFP的重組質(zhì)粒,通過(guò)觀察GFP熒光的強(qiáng)弱來(lái)體外研究GA6是否影響LC3的切割;通過(guò)GST pull-down實(shí)驗(yàn)探討GA6、LC3、Atg4B三者之間的定位關(guān)系,明確GA6影響LC3的切割的具體機(jī)制。為了研究脂化,我們構(gòu)建了GFP-LC3-Hela的穩(wěn)定細(xì)胞亞株,體內(nèi)通過(guò)流式細(xì)胞術(shù)檢測(cè)GFP的平均熒光強(qiáng)度(MFI,Median Fluorescent intensities)來(lái)反映LC3-II的形成情況;體外通過(guò)LC3-I與PE beads的結(jié)合情況來(lái)反映GA6對(duì)LC3-I的脂化的影響。在動(dòng)物水平方面,我們將野生型小鼠分為正常飼養(yǎng)組和自噬誘導(dǎo)組,分別進(jìn)行1000cGy劑量的(60)Co照射,統(tǒng)計(jì)生存曲線(xiàn)來(lái)研究自噬對(duì)小鼠放射敏感性的影響;將GA+/+組和GA6-/-組小鼠分別進(jìn)行1000 cGy劑量的(60)Co照射,統(tǒng)計(jì)生存曲線(xiàn)來(lái)研究GA6對(duì)小鼠放射敏感性的影響。結(jié)果:(1)LC3與GA6有直接相互作用。通過(guò)免疫共沉淀結(jié)合質(zhì)譜法、快速蛋白液相色譜法和免疫共沉淀法檢測(cè)到GA6與LC3有相互作用,通過(guò)GST pull-down確定LC3與GA6有直接相互作用,因此將研究中心定為L(zhǎng)C3。(2)GA6抑制自噬。免疫熒光顯示GA6敲低或者敲除均導(dǎo)致細(xì)胞質(zhì)中點(diǎn)狀LC3的形成增加,Western blot顯示GA6抑制LC3向LC3-II的轉(zhuǎn)化,透射電子顯微鏡顯示GA6抑制自噬體的形成。因?yàn)榱姿崧揉?CQD,Chloroquine Diphosphate Salt)處理后GA6對(duì)自噬的抑制作用更明顯,所以我們判斷GA6對(duì)自噬的抑制出現(xiàn)在自噬體與溶酶體融合之前,也就是自噬前體延長(zhǎng)成自噬體的過(guò)程。并通過(guò)雷帕霉素的處理發(fā)現(xiàn)GA6抑制自噬不依賴(lài)于mTOR通路。(3)GA6抑制Atg4B對(duì)LC3的切割。體內(nèi)實(shí)驗(yàn)表明,當(dāng)GA6過(guò)表達(dá)時(shí),LC3-PLA2的切割減少;體外實(shí)驗(yàn)表明,Atg4B單獨(dú)存在時(shí),GST-LC3-GFP的綠色熒光變?yōu)闊o(wú)色,說(shuō)明Atg4B可切割LC3;GA6、Atg4B同時(shí)存在時(shí),GST-LC3-GFP的綠色熒光變化不大,表明GA6抑制Atg4B對(duì)LC3的切割。又因?yàn)镚A6、Atg4B定位于LC3的同一片段,表明GA6與Atg4B競(jìng)爭(zhēng)性結(jié)合于LC3而導(dǎo)致GA6抑制Atg4B對(duì)LC3的切割。(4)GA6抑制LC3-I的脂化。體內(nèi)實(shí)驗(yàn)表明,當(dāng)GA6過(guò)表達(dá)時(shí),MFI of GFP-LC3-II減少;體外實(shí)驗(yàn)表明,當(dāng)有GA6存在時(shí),結(jié)合在PE beads上的LC3-I減少。(5)小鼠誘導(dǎo)自噬后表現(xiàn)為放射抵抗,GA6-/-小鼠表現(xiàn)為放射敏感。當(dāng)野生型正常飼養(yǎng)組和自噬誘導(dǎo)組小鼠經(jīng)過(guò)相同劑量的(60)Co照射后,生存曲線(xiàn)分析顯示,與正常飼養(yǎng)組相比,自噬誘導(dǎo)組小鼠死亡更慢;當(dāng)GA6+/+與GA6-/-小鼠經(jīng)過(guò)相同劑量的(60)Co照射后,生存曲線(xiàn)分析顯示,與GA6+/+小鼠相比,GA6-/-小鼠死亡更快。結(jié)論:發(fā)現(xiàn)了一個(gè)新的抑制自噬的分子——GA6。GA6通過(guò)與Atg4B競(jìng)爭(zhēng)性結(jié)合于LC3抑制Atg4B對(duì)pro-LC3的切割,從而抑制LC3-I的形成,GA6還抑制LC3-I的脂化。GA6由于抑制pro-LC3的切割和LC3-I的脂化導(dǎo)致自噬前體延長(zhǎng)障礙,從而抑制自噬。
[Abstract]:Objective: autophagy is a cellular pathway from eukaryotes to have highly conserved self consumption. Under conditions of starvation, cells through autophagy pathway to degrade its nucleotides, proteins and organelles of amino acids, fatty acids, carbohydrates and ATP, in order to maintain the normal metabolism of the cells to survive in the harsh environment autophagy, can remove protein misfolding and intracellular organelle damage, maintain cytoplasmic proteins and organelles at normal levels. The timely and appropriate occurrence of autophagy autophagy is a major guarantee the normal operation. Autophagy abnormalities and inflammation, tumor and neurodegenerative diseases and other diseases, the current target to autophagy 3- methyladenine and chloroquine drugs have been used for the treatment of cancer, in-depth understanding of autophagy can help guide the development of medical, medicine and clinical medicine, promote new treatment The strategy is produced. The main indexes of autophagy flow including pro-LC3 conversion to LC3-I, LC3-I to LC3-II conversion, the P62 degradation in autolysosome. Promote autophagy, cutting increased pro-LC3, increased lipid LC3-I, the degradation of P62 increased. The autophagy events include starting autophagy, nucleation and extension of autophagy the precursor film, autophagosome maturation, fusion of autophagosomes and lysosomes, degradation of phagosomes recycling. 5 amino acids involved in caspase pro-LC3 under the action of Atg4B was cut off at the end of the extended C autophagy precursor film (MKLSV) into LC3-I, LC3-I by Atg7, Atg3, Atg10, CO the role of Atg12-Atg5-Atg16L is to form lipid phosphatidylethanolamine (PE, Phosphatidylethanolamine) modified forms of LC3-II, and specifically located in the autophagy precursor film, which create the conditions for autophagosome maturation. But all this process is how to cut and fat suppression Is not clear, so this study intends to explore the effects of GA6 and GA6 in our laboratory found cancer protein in the cutting and fat in the process of autophagy. Content: This study mainly includes the following: 1) to investigate whether GA6 is related to autophagy. Screening the interaction between GA6 and protein. Verify.2 GA6) what is the effect of.GA6 on autophagy promotes autophagy or inhibition of autophagy, which performance can prove the effect of GA6 on LC3.3) cut.GA6 LC3 cleavage of what effect, the influence mechanism is what.4 GA6) effects on LC3-I lipid.GA6 of LC3-I fat what effect whether this effect because.5 caused the change of LC3-I) what is the effect of GA6 on autophagy phenotype in C57 mice. (60) combined with Co irradiation, GA6 analysis, the relationship between autophagy and radiosensitivity. Methods: to construct the GA6- / - breast cancer using CRISPR/Cas9 Technology Cell sublines ZR75-1 and GA6-/-C57 mice, and to determine the effective on siRNA low GA6, established the knockdown of GA6 cervical cancer cell line Hela. by immunization combined with mass spectrometry and fast protein liquid chromatography screening GA6 associated with autophagy protein co precipitation, and Co-IP and GA6, a judge whether it has interaction with GA6, then we use the GST pulldown experiment and ultimately determine which specific autophagy protein GA6 with direct interaction, and to determine the autophagy protein for the research center. The formation of immunofluorescence observation point LC3, the formation of application of transmission electron microscopy observation of autophagosomes and autolysosomes. The application of Western blot technique to detect LC3-I conversion to LC3-II. Through the treatment of chloroquine and rapamycin on autophagy, which link to explore the effects of GA6 on autophagy specific performance, and whether the MTOR pathway. In order to explore the effect of LC3 and GA6 cutting fat, we constructed the recombinant plasmid Myc-LC3-PLA2, in vivo effects of LC3 GA6 is cutting; construction of the recombinant plasmid GST-LC3-GFP, to study the in vitro effect of LC3 GA6 is cutting through the observation of GFP fluorescence intensity by GST pull-down; experimental study of GA6. LC3 Atg4B, relationship between the three, the specific mechanism of clear GA6 effect of LC3 cutting. In order to study the lipid, we construct GFP-LC3-Hela stable cell line in vivo, by the mean fluorescence intensity of GFP by flow cytometry (MFI Median, Fluorescent intensities) to reflect the formation of LC3-II in vitro by combining; LC3-I and PE beads to reflect the effect of GA6 on LC3-I resin. In the animal level, we will be the wild type mice were divided into normal diet group and autophagy induction group, respectively 1000cGy agent The amount of (60) Co irradiation, statistical survival curves to study autophagy of mice Radiosensitivity; GA+/+ group and GA6-/- group were respectively 1000 doses of cGy (60) Co irradiation, the statistical survival curve to study the influence of GA6 on the radiosensitivity of mice. Results: (1) LC3 and GA6 have each other directly the role of the immune co precipitation method combined with mass spectrometry, fast protein liquid chromatography method to detect the GA6 and LC3 interaction and co immunoprecipitation, LC3 and GA6 have a direct interaction with GST pull-down, the Research Center for LC3. (2) GA6 inhibited autophagy. Immunofluorescence showed that GA6 knockdown or knockout were led to the formation of cytoplasmic punctate LC3 increases, Western blot showed that GA6 inhibited LC3 to LC3-II conversion, transmission electron microscopy showed the formation of autophagosomes. Because of the inhibition of GA6 (CQD Chloroquine Diphosphate, chloroquine phosphate Salt) after the treatment of GA6 on autophagy The inhibitory effect is more obvious, so we determine the inhibition of GA6 on autophagy occurs before and lysosome fusion, is also the precursor to extend into the process of autophagy autophagy by rapamycin treatment. It was found that GA6 inhibited autophagy is not dependent on mTOR pathway. (3) GA6 inhibited Atg4B LC3 cleavage of the in vivo experiments show. When, over expression of GA6, LC3-PLA2 cutting decreased; the in vitro experiments showed that Atg4B alone, the green fluorescence of GST-LC3-GFP becomes colorless, Atg4B LC3 GA6, Atg4B cutting; existing at the same time, the green fluorescence of GST-LC3-GFP changed little, suggesting that GA6 inhibits LC3 cleavage of Atg4B. Because GA6, Atg4B positioning the same segment to LC3, GA6 inhibited Atg4B LC3 cleavage of which showed that GA6 and Atg4B competitive binding to LC3. (4) GA6 inhibits LC3-I lipidation. In vivo experiments showed that the overexpression of GA6, MFI of GFP-LC3-II was decreased; in vitro. The results show that, when there is GA6, with PE beads in LC3-I decreased. (5) mice showed resistance to radiation induced autophagy, GA6-/- mice exhibited radiation sensitive. When the wild type normal feeding group and autophagy induced group of mice after the same dose (60 Co) after irradiation, the survival curve analysis showed that compared with the normal diet group, slower death induced autophagy mice; when GA6+/+ and GA6-/- mice after the same dose (60 Co) after irradiation, the survival curve analysis showed that, compared with GA6+/+ mice, GA6-/- mice died faster. Conclusion: the discovery of a new molecular GA6.GA6 inhibition of autophagy by binding to the Atg4B competition in LC3 suppressed Atg4B cleavage of pro-LC3, thus inhibiting the formation of LC3-I, GA6.GA6 LC3-I also inhibited lipid lipid due to inhibition of pro-LC3 cutting and LC3-I lead to autophagy precursor to inhibit autophagy. Extend the obstacles

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q23
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本文編號(hào)：1625257