本文選題：MTA1 + 外泌體��； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的探討食管癌外泌體介導(dǎo)的腫瘤細(xì)胞之間的惡性表型傳遞以及MTA1對(duì)食管癌細(xì)胞外泌體特性及蛋白成分影響。方法采用差速超速離心法提取KYSE410細(xì)胞外泌體,通過(guò)透射電鏡及免疫印跡法(Western blotting)觀察所獲得的外泌體的形態(tài)及其標(biāo)志物;共聚焦顯微鏡觀察KYSE410、KYSE510和YES2細(xì)胞攝取熒光染料標(biāo)記的KYSE410外泌體;分別采用transwell實(shí)驗(yàn)、劃痕愈合實(shí)驗(yàn)、細(xì)胞增殖實(shí)驗(yàn)、克隆形成實(shí)驗(yàn)檢驗(yàn)KYSE410細(xì)胞外泌體對(duì)3種食管癌細(xì)胞遷移、侵襲、增殖和克隆形成的影響;免疫印跡法檢測(cè)Wnt/β-catenin和PI3K/Akt信號(hào)通路相關(guān)蛋白的變化。分別對(duì)MTA1共表達(dá)基因和MTA1 Co-IP質(zhì)譜相互作用蛋白作聚類分析;Nanosight檢測(cè)不同MTA1表達(dá)水平的食管癌細(xì)胞分泌的外泌體粒徑大小;Label-free質(zhì)譜方法檢測(cè)不同MTA1表達(dá)水平的食管癌細(xì)胞的外泌體蛋白并對(duì)差異蛋白作GO功能聚類分析;通過(guò)CCLE數(shù)據(jù)庫(kù)和cBioPortal平臺(tái)數(shù)據(jù)分析MTA1和目標(biāo)差異蛋白在組織、細(xì)胞水平的相關(guān)性;分析目標(biāo)差異蛋白與臨床病理特征的相關(guān)性。結(jié)果透射電鏡可以觀察到具有膜結(jié)構(gòu)的外泌體;免疫印跡方法能夠檢測(cè)到外泌體標(biāo)志蛋白CD63和CD81;3種細(xì)胞均能夠成功攝取熒光染料標(biāo)記的KYSE410外泌體;3種食管癌細(xì)胞的遷移及侵襲能力從大到小依次為KYSE410、KYSE510、YES2,KYSE410外泌體能夠促進(jìn)3種食管癌細(xì)胞遷移、侵襲及克隆形成,但不影響3種食管癌細(xì)胞的增殖;外泌體處理能夠增強(qiáng)3種食管癌細(xì)胞中β-catenin和p-Akt的表達(dá)。MTA1共表達(dá)基因聚類分析顯示MTA1影響囊泡組織與生物合成;MTA1 Co-IP質(zhì)譜相互作用蛋白聚類分析顯示絕大多數(shù)MTA1相互作用蛋白在外泌體中有定位;不同MTA1表達(dá)水平的KYSE410細(xì)胞和KYSE450細(xì)胞分泌的外泌體的大小和數(shù)量沒(méi)有顯著差異;首次鑒定出25個(gè)新的外泌體蛋白成分;CytoScape軟件分析兩組外泌體蛋白連接度最高的節(jié)點(diǎn)均在UBC蛋白;對(duì)照/敲除MTA1的KYSE410細(xì)胞獲得外泌體蛋白和對(duì)照/過(guò)表達(dá)KYSE450細(xì)胞獲得的外泌體蛋白都主要定位在胞漿、細(xì)胞核、溶酶體、質(zhì)膜等部位,主要的分子功能包括催化活性、分子伴侶活性、細(xì)胞骨架蛋白結(jié)合、GTPase活性等,主要影響的生物學(xué)進(jìn)程包括蛋白代謝、能量通路、細(xì)胞生長(zhǎng)和維持、免疫反應(yīng)等;廣泛參與整聯(lián)蛋白家族細(xì)胞表面相互作用、β1整聯(lián)蛋白細(xì)胞表面相互作用、蛋白多糖syndecan介導(dǎo)的信號(hào)事件等;對(duì)照/敲除MTA1的KYSE410細(xì)胞和對(duì)照/過(guò)表達(dá)KYSE450細(xì)胞的外泌體內(nèi)MTA1調(diào)控的差異蛋白共同參與大分子代謝負(fù)調(diào)控、磷酸鹽代謝、大分子分解代謝等生物學(xué)進(jìn)程;MYH9、SLC7A5和CCT4均與MTA1在組織水平有中等強(qiáng)度相關(guān)性;MYH9、CCT4和食管癌病人T分期、臨床分期有相關(guān)性;SLC7A5、CCT4高表達(dá)的食管癌病人總生存時(shí)間更短。結(jié)論高侵襲性食管癌細(xì)胞KYSE410來(lái)源的外泌體能夠促進(jìn)自身以及食管癌KYSE510、YES2細(xì)胞的遷移和侵襲、克隆形成,其可能是通過(guò)激活Wnt/β-catenin和PI3K/Akt信號(hào)通路發(fā)揮上述作用的。聚類分析結(jié)果表明MTA1可能參與外泌體調(diào)控;MTA1不影響食管癌細(xì)胞分泌外泌體的大小和總量;但MTA1影響食管癌細(xì)胞外泌體的蛋白質(zhì)組成;關(guān)鍵的差異蛋白MYH9、SLC7A5、CCT4可能作為食管癌的預(yù)后評(píng)價(jià)指標(biāo)。
[Abstract]:Objective to investigate the malignant phenotype transmission between tumor cells mediated by esophageal carcinoma and the effects of MTA1 on the extracellular secretory and protein components of esophageal cancer cells. Methods the extracellular secretory of KYSE410 cells was extracted by differential velocity centrifugation, and the morphology of the exosecrete obtained by transmission electron microscopy and immunoblotting (Western blotting) was observed and the morphology of the exosecrete was observed by transmission electron microscopy and immunoblotting (blotting). Its markers; confocal microscopy (confocal microscopy) to observe the KYSE410 Exocyst marked by KYSE410, KYSE510 and YES2 cells with fluorescent dye; Transwell experiment, scratch healing experiment, cell proliferation experiment, and clone formation test were used to test the effect of KYSE410 cell exocrine on the migration, invasion, proliferation and cloning of 3 kinds of esophageal cancer cells. The changes of Wnt/ beta -catenin and PI3K/Akt signal pathway related proteins were detected by trace method. The MTA1 coexpression gene and MTA1 Co-IP mass interaction protein were cluster analysis respectively; Nanosight was used to detect the outer secretory particle size of esophageal cancer cells with different MTA1 expression levels, and the Label-free spectrum method was used to detect the esophagus with different MTA1 expression levels. The exocrine protein of cancer cells was analyzed by GO function cluster analysis, and the correlation of MTA1 and target differential protein in tissue and cell level was analyzed by CCLE database and cBioPortal platform data, and the correlation between target differential protein and clinicopathological features was analyzed. The immunoblotting method can detect the exosecreting protein CD63 and CD81, and 3 kinds of cells can successfully absorb the KYSE410 exote marked by fluorescent dyes; the migration and invasion ability of the 3 esophageal cancer cells from large to small are KYSE410, KYSE510, YES2, and the KYSE410 exote can promote the migration, invasion and cloning of 3 kinds of esophageal cancer cells. The proliferation of 3 esophageal cancer cells was not affected; exosecretory treatment enhanced the expression of.MTA1 co expression genes in 3 esophageal cancer cells and.MTA1 co expression gene cluster analysis showed that MTA1 affects vesicle tissue and biosynthesis; MTA1 Co-IP mass spectrometry interaction protein cluster analysis showed that most of MTA1 interacting proteins were determined in exosecrete. There was no significant difference in the size and quantity of exocrine secreted by KYSE410 and KYSE450 cells at different levels of MTA1 expression; 25 new exocrine proteins were identified for the first time; CytoScape software analyzed the nodes with the highest degree of exocytosis of the two groups in UBC protein; and the exocrine protein was obtained for KYSE410 cells that were illuminated / knocked out of MTA1. The exosecreting proteins obtained from the controlled / overexpressed KYSE450 cells are mainly located in the cytoplasm, nucleus, lysosome, plasma membrane and other parts. The main molecular functions include catalytic activity, molecular chaperone activity, cytoskeleton binding, and GTPase activity. The main effects of biological processes include protein metabolism, energy pathway, cell growth and dimension. Holding, immune responses, and so on; widely involved in the cell surface interaction of the integrin family, the surface interaction of the beta 1 integrin cells, the signaling events mediated by the proteoglycan syndecan, and the negative modulation of the MTA1 KYSE410 cells and the control / overexpressed KYSE450 cells in the MTA1 regulation of the exocrine KYSE450 cells. Biological processes such as control, phosphate metabolism, macromolecular catabolism and other biological processes; MYH9, SLC7A5 and CCT4 have moderate correlation with MTA1 at the tissue level; MYH9, CCT4 and esophageal cancer patients have a correlation of T staging and clinical staging; SLC7A5 and CCT4 high expression of esophageal cancer have shorter total survival time. Conclusion high invasive esophageal cancer cells are of KYSE410 origin. Exosecrete can promote the migration and invasion of KYSE510 and YES2 cells in the carcinoma of the esophagus and the formation of the clone formation, which may be played by activating the Wnt/ beta -catenin and PI3K/Akt signaling pathway. Cluster analysis results show that MTA1 may be involved in exocrine regulation; MTA1 does not affect the size and total amount of exocrine secreted by cancer cells; but MTA 1 the protein composition of exocrine cells in esophageal cancer may be affected. The key differentially expressed proteins MYH9, SLC7A5 and CCT4 may be used as prognostic indicators for esophageal cancer.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1

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