LMP1第三个功能活性区对鼻咽癌干细胞SP18生长的影响及机制

发布时间：2018-01-16 20:33

本文关键词：LMP1第三个功能活性区对鼻咽癌干细胞SP18生长的影响及机制　出处：《南华大学》2011年硕士论文　论文类型：学位论文

【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。本研究探讨LMP1第三个功能活性区对鼻咽癌干细胞SP18生长的影响及机制，为揭示EBV的致瘤机理提供实验依据。方法：收集RV-LMP1和RV-LMP1△232-351逆病毒，分别感染鼻咽癌干细胞SP18，G418筛选后汇合克隆，建立稳定表达LMP1及CTAR3缺失突变型LMP1（LMP1△232-351）的SP18-LMP1细胞和SP18-LMP1△232-351细胞系。然后采用细胞生长曲线、软琼脂集落形成实验和平皿克隆形成实验观察LMP1△232-351对SP18生长的影响，其次选用流式细胞仪检测细胞周期，并计算细胞增殖指数。另外，，利用基因芯片检测LMP1及LMP1△232-351对SP18细胞生长影响的差异表达基因，RT-PCR验证SP18-LMP1细胞和SP18-LMP1△232-351细胞中部分基因的表达。结果：1、细胞生长曲线、软琼脂集落和平皿克隆结果显示，SP18-LMP1△232-351细胞较SP18-LMP1细胞生长速度减慢，集落数目减少，体积变小（n=3，p0.01）；2、流式细胞仪检测结果显示，SP18-LMP1△232-351细胞较SP18-LMP1细胞增殖指数降低（n=3，p0.01）。3、基因芯片检测结果显示，在SP18-LMP1细胞和SP18-LMP1△232-351细胞间共测出428个差异表达基因，其中与细胞生长增殖相关基因135个（上调基因59个，下调基因76个）。6、RT-PCR验证部分基因的表达结果与基因芯片检测结果基本一致。结论： 1、LMP1-CTAR3是其促SP18细胞生长的重要功能活性部位。 2、LMP1-CTAR3通过增加细胞的增殖指数，促SP18细胞的生长增殖。 3、LMP1-CTAR3通过影响IL1A和Wnt6基因的表达，调节SP18细胞的生长。
[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.
【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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