本文選題：mP19　切入點(diǎn)：MARVELD1　出處：《哈爾濱工業(yè)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 本課題組前期實(shí)驗(yàn)證實(shí)MARVELD1是一個(gè)在多種腫瘤細(xì)胞中呈現(xiàn)低表達(dá)的候選抑癌基因,其功能研究有待進(jìn)一步深入。同源比對(duì)分析發(fā)現(xiàn)來(lái)自小鼠的mP19與人源MARVELD1氨基酸序列同源性達(dá)88%,且結(jié)構(gòu)域高度保守。因此,本課題通過(guò)生化和細(xì)胞生物學(xué)技術(shù)探討mP19的基本功能,旨在闡明mP19和MARVELD1的功能異同,為后續(xù)建立小鼠模型提供一定參考。本課題中通過(guò)生物信息學(xué)方法,我們推斷mP19及其種屬同源分子僅在脊椎動(dòng)物中表達(dá)且在高等哺乳類中呈現(xiàn)高度保守性。mP19具有四個(gè)潛在的蛋白激酶C(PKC)磷酸化位點(diǎn)(Thr15, Ser19, Thr51, Thr120),而人源MARVELD1僅存在一個(gè)(Thr51),我們采用免疫沉淀技術(shù)及Western Blot方法檢測(cè)證實(shí)mP19存在可被磷酸化的蘇氨酸,而Thr51因其在人鼠序列中的高度保守性被認(rèn)為是mP19的PKC磷酸化關(guān)鍵蘇氨酸。我們采用免疫熒光技術(shù),在共聚焦顯微鏡下觀察到mP19呈現(xiàn)周期依賴性細(xì)胞定位:間期時(shí)定位于細(xì)胞核及細(xì)胞核周,分裂期時(shí)與紡錘體微管及胞質(zhì)分裂器有明顯共定位。佛波酯醇(PMA)激活PKC能促進(jìn)mP19的核轉(zhuǎn)位。通過(guò)GST-pull down和免疫共沉淀技術(shù),我們證實(shí)mP19與α-tubulin存在相互作用,且秋水仙素能促進(jìn)mP19向細(xì)胞膜轉(zhuǎn)位。我們利用相差顯微鏡、共聚焦顯微鏡及掃描電鏡從不同角度觀察到過(guò)表達(dá)mP19抑制細(xì)胞微絲骨架組裝、促進(jìn)細(xì)胞表面超微結(jié)構(gòu)和絲狀偽足的形成。此外,我們證實(shí)在NIH3T3細(xì)胞中過(guò)表達(dá)mP19能明顯抑制細(xì)胞增殖、造成細(xì)胞G1期阻滯、抑制細(xì)胞遷移運(yùn)動(dòng)。
[Abstract]:Our previous experiments confirmed that MARVELD1 is a low expression in many tumor cells candidate tumor suppressor gene, its function needs further study. Homology analysis found that mouse mP19 and human MARVELD1 amino acid sequence homology of 88%, and the domain is highly conserved. Therefore, the basic function of this subject through study of Biochemistry and cell biology, technology of mP19, mP19 and MARVELD1 to elucidate the functional similarities and differences, to provide a reference for the subsequent establishment of mouse model. Through bioinformatics in this topic studies, we conclude that mP19 and a homologous molecule expressed only in vertebrates and in mammals in highly conserved.MP19 has had four potential protein kinase C (PKC) phosphorylation sites (Thr15, Ser19, Thr51, Thr120), and there is only one source of MARVELD1 (Thr51), we used immunoprecipitation and Western Blot assay confirmed the existence of mP19 can be phosphorylated threonine, and Thr51 because of its highly conserved sequence in the rat was found to be in mP19 PKC phosphorylation of key threonine. We used immunofluorescence technique, mP19 were observed by confocal microscopy showed cell cycle dependent location: located in the nucleus and the interphase nuclear week, mitotic spindle microtubules and cytokinesis and has obvious colocalization. Phorbol ester alcohol (PMA) activation of PKC can promote the nuclear translocation of mP19. By GST-pull down and immunoprecipitation, we demonstrate that mP19 interacts with the alpha -tubulin, and colchicine can promote mP19 to cell membrane translocation. We use difference microscopy, confocal microscopy and scanning electron microscopy from different angles observed that overexpression of mP19 inhibits the actin cytoskeleton assembly, promote the formation of cell surface ultrastructure and filopodia. In addition, we confirm that overexpression of mP19 in NIH3T3 cells can significantly inhibit cell proliferation, cause G1 phase arrest and inhibit cell migration.

【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346;Q25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王山;肖晟;赫杰;韓放;李鈺;;核蛋白MARVELD1與核質(zhì)轉(zhuǎn)運(yùn)受體蛋白Importin β1相互作用的鑒定[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);2009年08期

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本文編號(hào)：1586277