本文關(guān)鍵詞：LMP1第三個功能活性區(qū)對鼻咽癌干細胞SP18生長的影響及機制　出處：《南華大學》2011年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 鼻咽癌干細胞 EB病毒 LMP1 SP18細胞 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第三個功能活性區(qū)對鼻咽癌干細胞SP18生長的影響及機制，為揭示EBV的致瘤機理提供實驗依據(jù)。 方法：收集RV-LMP1和RV-LMP1△232-351逆病毒，分別感染鼻咽癌干細胞SP18，G418篩選后匯合克隆，建立穩(wěn)定表達LMP1及CTAR3缺失突變型LMP1（LMP1△232-351）的SP18-LMP1細胞和SP18-LMP1△232-351細胞系。然后采用細胞生長曲線、軟瓊脂集落形成實驗和平皿克隆形成實驗觀察LMP1△232-351對SP18生長的影響，其次選用流式細胞儀檢測細胞周期，并計算細胞增殖指數(shù)。另外，，利用基因芯片檢測LMP1及LMP1△232-351對SP18細胞生長影響的差異表達基因，RT-PCR驗證SP18-LMP1細胞和SP18-LMP1△232-351細胞中部分基因的表達。 結(jié)果：1、細胞生長曲線、軟瓊脂集落和平皿克隆結(jié)果顯示，SP18-LMP1△232-351細胞較SP18-LMP1細胞生長速度減慢，集落數(shù)目減少，體積變�。╪=3，p0.01）；2、流式細胞儀檢測結(jié)果顯示，SP18-LMP1△232-351細胞較SP18-LMP1細胞增殖指數(shù)降低（n=3，p0.01）。3、基因芯片檢測結(jié)果顯示，在SP18-LMP1細胞和SP18-LMP1△232-351細胞間共測出428個差異表達基因，其中與細胞生長增殖相關(guān)基因135個（上調(diào)基因59個，下調(diào)基因76個）。6、RT-PCR驗證部分基因的表達結(jié)果與基因芯片檢測結(jié)果基本一致。 結(jié)論： 1、LMP1-CTAR3是其促SP18細胞生長的重要功能活性部位。 2、LMP1-CTAR3通過增加細胞的增殖指數(shù)，促SP18細胞的生長增殖。 3、LMP1-CTAR3通過影響IL1A和Wnt6基因的表達，調(diào)節(jié)SP18細胞的生長。
[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

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