本文選題：鼻咽癌干細胞 + EB病毒��； 參考：《南華大學》2011年碩士論文

【摘要】：目的：鼻咽癌（NPC）的發(fā)病與EB病毒感染密切相關(guān)，EB病毒編碼的潛伏性膜蛋白1（LMP1）是其重要致瘤蛋白。本研究旨在探討LMP1羧基末端CTAR2的TRADD活性區(qū)在鼻咽癌干細胞SP18增殖中的作用及機制，為揭示EBV的致瘤機理提供實驗依據(jù)。 方法：培養(yǎng)產(chǎn)RV-LMP1和RV-LMP1TRADD逆病毒的PA317細胞，，收集細胞上清，分別感染鼻咽癌干細胞SP18，經(jīng)G418篩選后融合克隆，免疫細胞化學染色檢測LMP1和LMP1TRADD蛋白的表達，建立表達LMP1及CTAR2突變LMP1（LMP1TRADD）的SP18細胞系，即SP18-LMP1細胞和SP18-LMP1TRADD細胞。然后繪制細胞生長曲線、平皿克隆實驗、軟瓊脂集落實驗和流式細胞術(shù)觀察LMP1TRADD對SP18增殖的影響。然后，采用基因芯片檢測SP18-LMP1細胞和SP18-LMP1TRADD細胞間的差異表達基因，并選用RT-PCR驗證部分基因的表達。 結(jié)果：1、免疫細胞化學染色結(jié)果顯示，建立的SP18-LMP1細胞和SP18-LMP1TRADD細胞均在細胞膜和漿中表達LMP1蛋白；2、繪制的細胞生長曲線結(jié)果顯示，SP18-LMP1細胞生長速度較SP18-LMP1TRADD細胞快（n=3，p0.01）；3、軟瓊脂集落和平皿克隆結(jié)果顯示，SP18-LMP1細胞較SP18-LMP1TRADD細胞形成的集落數(shù)多，體積大（n=3，p0.01）；4、流式細胞術(shù)檢測結(jié)果顯示，SP18-LMP1細胞S期和G2期細胞比例較SP18-LMP1TRADD細胞多，細胞增殖指數(shù)高（n=3，p0.01）。5、經(jīng)基因芯片共檢測出428個差異表達基因，其中與細胞增殖相關(guān)基因76個（上調(diào)基因38個，下調(diào)基因38個）。6、RT-PCR驗證部分基因的表達情況與基因芯片檢測結(jié)果基本一致。 結(jié)論： 1、TRADD活性區(qū)是LMP1影響SP18細胞增殖的重要功能活性位點。 2、TRADD活性區(qū)通過提高S期和G2期細胞比例，增加細胞的增殖指數(shù)，影響SP18細胞的增殖。 3、LMP1羧基末端CTAR2的TRADD活性區(qū)可能通過影響CYP1A1和NGFR等基因的表達，調(diào)節(jié)SP18細胞的增殖。
[Abstract]:Objective: the pathogenesis of nasopharyngeal carcinoma (NPC) is closely related to Epstein-Barr virus (EBV) infection. Epstein-Barr virus (EBV) encoded latent membrane protein (LMP1) is an important tumorigenic protein. The purpose of this study was to investigate the role and mechanism of TRADD active region of CTAR2 at the carboxyl end of LMP1 in the proliferation of nasopharyngeal carcinoma stem cells SP18. Methods: PA317 cells producing RV-LMP1 and RV-LMP1TRADD retrovirus were cultured and supernatants were collected. SP18 cells were infected with nasopharyngeal carcinoma stem cells SP18 and fused with G418. The expression of LMP1 and LMP1TRADD protein was detected by immunocytochemical staining. SP18 cell lines expressing LMP1 and CTAR2 mutation LMP1 / LMP1TRADDD) were established, namely SP18-LMP1 cells and SP18-LMP1TRADD cells. Then the cell growth curve, plate clone assay, soft Agar colony assay and flow cytometry were used to observe the effect of LMP1TRADD on the proliferation of SP18. Then the differentially expressed genes between SP18-LMP1 cells and SP18-LMP1TRADD cells were detected by gene chip, and some genes were detected by RT-PCR. Both SP18-LMP1 cells and SP18-LMP1TRADD cells expressed LMP1 protein 2 in cell membrane and plasma. The results of cell growth curve showed that SP18-LMP1 cells grew faster than SP18-LMP1TRADD cells and SP18-LMP1TRADD cells grew faster than SP18-LMP1TRADD cells. The results of soft Agar colony and pan cloning showed that SP18-LMP1 cells were finer than SP18-LMP1TRADD cells. The number of colonies formed by cell is many, The results of flow cytometry showed that the proportion of S phase and G2 phase of SP18-LMP1 cells was more than that of SP18-LMP1TRADD cells, and the proliferation index of SP18-LMP1TRADD cells was higher than that of SP18-LMP1TRADD cells. A total of 428 differentially expressed genes were detected by gene chip. Among them, 76 genes were associated with cell proliferation (38 up-regulated genes). Conclusion: 1 the active region of TRADD is an important functional active site of LMP1 to affect the proliferation of SP18 cells. 2 the active region of TRADD can increase the proportion of cells in S phase and G2 phase. The TRADD active region of CTAR2 at the carboxyl end of LMP1 may regulate the proliferation of SP18 cells by affecting the expression of CYP1A1 and NGFR genes.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R739.63

【參考文獻】

相關(guān)期刊論文 前8條

1 張志偉;賀智敏;周敏;張瓊;余艷輝;陳主初;;NF-κB介導的EBV-LMP1在Rat-1細胞轉(zhuǎn)化和成瘤中的作用[J];癌癥;2007年02期

2 張志偉;賀智敏;周敏;丁渭;余艷輝;陳主初;;EBV-LMP1促CNE2細胞遷移的作用機制[J];中南大學學報(醫(yī)學版);2006年04期

3 張志偉;張瓊;余艷輝;歐陽詠梅;賀智敏;;EB病毒LMP1-CTAR_3對NP69細胞增殖和蛋白質(zhì)表達的影響[J];生物化學與生物物理進展;2009年05期

4 賀智敏,陳主初;EB病毒潛伏性膜蛋白CTAR-2突變體的構(gòu)建及功能分析[J];生物化學與生物物理學報;2003年03期

5 張志偉;張瓊;余艷輝;歐陽詠梅;賀智敏;;Epstein-Barr病毒潛伏性膜蛋白1 CTAR_3缺失突變體的構(gòu)建與功能分析[J];微生物學報;2008年10期

6 賀智敏;張志偉;楊芳;余艷輝;歐陽詠梅;陳主初;;EB病毒潛伏性膜蛋白1促原代MEF細胞永生化的作用及機制[J];Virologica Sinica;2006年01期

7 王承興,李曉艷,顧煥華,鄧錫云,曹亞;EB病毒LMP1在鼻咽癌細胞系中上調(diào)EGFR表達[J];中華腫瘤雜志;2001年04期

8 陶怡,張學光;EB病毒編碼潛伏性膜蛋白1的信號傳導和生物學特性及與CD40的相關(guān)性[J];中國醫(yī)學科學院學報;2004年05期

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