本文關(guān)鍵詞：LMP1第三個(gè)功能活性區(qū)對(duì)鼻咽癌干細(xì)胞SP18生長的影響及機(jī)制　出處：《南華大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鼻咽癌干細(xì)胞 EB病毒 LMP1 SP18細(xì)胞 基因芯片 生長增殖

【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。本研究探討LMP1第三個(gè)功能活性區(qū)對(duì)鼻咽癌干細(xì)胞SP18生長的影響及機(jī)制，為揭示EBV的致瘤機(jī)理提供實(shí)驗(yàn)依據(jù)。 方法：收集RV-LMP1和RV-LMP1△232-351逆病毒，分別感染鼻咽癌干細(xì)胞SP18，G418篩選后匯合克隆，建立穩(wěn)定表達(dá)LMP1及CTAR3缺失突變型LMP1（LMP1△232-351）的SP18-LMP1細(xì)胞和SP18-LMP1△232-351細(xì)胞系。然后采用細(xì)胞生長曲線、軟瓊脂集落形成實(shí)驗(yàn)和平皿克隆形成實(shí)驗(yàn)觀察LMP1△232-351對(duì)SP18生長的影響，其次選用流式細(xì)胞儀檢測(cè)細(xì)胞周期，并計(jì)算細(xì)胞增殖指數(shù)。另外，，利用基因芯片檢測(cè)LMP1及LMP1△232-351對(duì)SP18細(xì)胞生長影響的差異表達(dá)基因，RT-PCR驗(yàn)證SP18-LMP1細(xì)胞和SP18-LMP1△232-351細(xì)胞中部分基因的表達(dá)。 結(jié)果：1、細(xì)胞生長曲線、軟瓊脂集落和平皿克隆結(jié)果顯示，SP18-LMP1△232-351細(xì)胞較SP18-LMP1細(xì)胞生長速度減慢，集落數(shù)目減少，體積變�。╪=3，p0.01）；2、流式細(xì)胞儀檢測(cè)結(jié)果顯示，SP18-LMP1△232-351細(xì)胞較SP18-LMP1細(xì)胞增殖指數(shù)降低（n=3，p0.01）。3、基因芯片檢測(cè)結(jié)果顯示，在SP18-LMP1細(xì)胞和SP18-LMP1△232-351細(xì)胞間共測(cè)出428個(gè)差異表達(dá)基因，其中與細(xì)胞生長增殖相關(guān)基因135個(gè)（上調(diào)基因59個(gè)，下調(diào)基因76個(gè)）。6、RT-PCR驗(yàn)證部分基因的表達(dá)結(jié)果與基因芯片檢測(cè)結(jié)果基本一致。 結(jié)論： 1、LMP1-CTAR3是其促SP18細(xì)胞生長的重要功能活性部位。 2、LMP1-CTAR3通過增加細(xì)胞的增殖指數(shù)，促SP18細(xì)胞的生長增殖。 3、LMP1-CTAR3通過影響IL1A和Wnt6基因的表達(dá)，調(diào)節(jié)SP18細(xì)胞的生長。
[Abstract]:Objective: the incidence 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. In this study, we investigated the effect of the third active region of LMP1 on the growth of nasopharyngeal carcinoma stem cells (SP18) and its mechanism. To provide experimental basis for revealing the tumorigenic mechanism of EBV. Methods: RV-LMP1 and RV-LMP1 232-351 retrovirus were collected and infected with nasopharyngeal carcinoma stem cells SP18 G418 respectively. Establishment of stable expression of LMP1 and CTAR3 deletion Mutant LMP1(LMP1 232-351. SP18-LMP1 cells and SP18-LMP1 232-351 cell lines were obtained. Then the cell growth curve was used. The effect of LMP1 232-351 on the growth of SP18 was observed by the soft Agar colony forming assay and the cell cycle was detected by flow cytometry. The cell proliferation index was calculated. In addition, the differentially expressed genes of LMP1 and LMP1 232-351 on the growth of SP18 cells were detected by gene microarray. RT-PCR was used to verify the expression of some genes in SP18-LMP1 cells and SP18-LMP1 232-351 cells. Results the cell growth curve and soft Agar colony cell clone showed that the growth rate of SP18-LMP1 232-351 cells was slower than that of SP18-LMP1 cells. The number of colonies decreased and the volume became smaller. 2. The results of flow cytometry showed that the proliferation index of SP18-LMP1 232-351 cells was lower than that of SP18-LMP1 cells. A total of 428 differentially expressed genes were detected between SP18-LMP1 cells and SP18-LMP1 232-351 cells. Among them, 135 genes related to cell growth and proliferation (59 up-regulated genes and 76 down-regulated genes) were confirmed by RT-PCR. Conclusion: 1 LMP1-CTAR3 is an important functional active site of LMP1-CTAR3 to promote the growth of SP18 cells. 2LMP1-CTAR3 promoted the growth and proliferation of SP18 cells by increasing cell proliferation index. 3 LMP1-CTAR3 regulates the growth of SP18 cells by affecting the expression of IL1A and Wnt6 genes.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 張志偉;賀智敏;周敏;張瓊;余艷輝;陳主初;;NF-κB介導(dǎo)的EBV-LMP1在Rat-1細(xì)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(xì)胞遷移的作用機(jī)制[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)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(duì)NP69細(xì)胞增殖和蛋白質(zhì)表達(dá)的影響[J];生物化學(xué)與生物物理進(jìn)展;2009年05期

4 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(xué)報(bào);2008年10期

5 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(xì)胞永生化的作用及機(jī)制[J];Virologica Sinica;2006年01期

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