基因芯片分析EB病毒LMP1诱导鼻咽癌干细胞增殖的机制

发布时间：2018-06-11 14:40

本文选题：鼻咽癌干细胞 + EB病毒　；参考：《南华大学》2011年硕士论文

【摘要】：目的：鼻咽癌（NPC）的发病与EB病毒感染密切相关，EB病毒编码的潜伏性膜蛋白1（LMP1）是其重要致瘤蛋白。本研究旨在探讨LMP1羧基末端CTAR2的TRADD活性区在鼻咽癌干细胞SP18增殖中的作用及机制，为揭示EBV的致瘤机理提供实验依据。方法：培养产RV-LMP1和RV-LMP1TRADD逆病毒的PA317细胞，，收集细胞上清，分别感染鼻咽癌干细胞SP18，经G418筛选后融合克隆，免疫细胞化学染色检测LMP1和LMP1TRADD蛋白的表达，建立表达LMP1及CTAR2突变LMP1（LMP1TRADD）的SP18细胞系，即SP18-LMP1细胞和SP18-LMP1TRADD细胞。然后绘制细胞生长曲线、平皿克隆实验、软琼脂集落实验和流式细胞术观察LMP1TRADD对SP18增殖的影响。然后，采用基因芯片检测SP18-LMP1细胞和SP18-LMP1TRADD细胞间的差异表达基因，并选用RT-PCR验证部分基因的表达。结果：1、免疫细胞化学染色结果显示，建立的SP18-LMP1细胞和SP18-LMP1TRADD细胞均在细胞膜和浆中表达LMP1蛋白；2、绘制的细胞生长曲线结果显示，SP18-LMP1细胞生长速度较SP18-LMP1TRADD细胞快（n=3，p0.01）；3、软琼脂集落和平皿克隆结果显示，SP18-LMP1细胞较SP18-LMP1TRADD细胞形成的集落数多，体积大（n=3，p0.01）；4、流式细胞术检测结果显示，SP18-LMP1细胞S期和G2期细胞比例较SP18-LMP1TRADD细胞多，细胞增殖指数高（n=3，p0.01）。5、经基因芯片共检测出428个差异表达基因，其中与细胞增殖相关基因76个（上调基因38个，下调基因38个）。6、RT-PCR验证部分基因的表达情况与基因芯片检测结果基本一致。结论： 1、TRADD活性区是LMP1影响SP18细胞增殖的重要功能活性位点。 2、TRADD活性区通过提高S期和G2期细胞比例，增加细胞的增殖指数，影响SP18细胞的增殖。 3、LMP1羧基末端CTAR2的TRADD活性区可能通过影响CYP1A1和NGFR等基因的表达，调节SP18细胞的增殖。
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

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