鼻咽癌上皮细胞EB病毒感染模型的构建及其相关基因的筛选
发布时间：2018-11-18 15:55
【摘要】：目的:通过EBV颗粒直接感染以及与携带EBV的霍奇金淋巴瘤细胞KMH2共培养两种方式,筛选并建立适宜的EBV感染鼻咽癌细胞系模型。进而通过该模型利用转录组测序技术检测分析鼻咽癌细胞体外感染EBV前后时间序列的差异表达基因,为进一步探讨EBV感染上皮细胞的分子机制奠定基础。方法:通过AGS-EBV-GFP细胞制备EBV-GFP,并建立EBV阳性的KMH2细胞株,用制备的EBV颗粒直接感染鼻咽癌细胞及用携带EBV的KMH2细胞与鼻咽癌细胞直接接触共培养方式,筛选适宜的鼻咽癌细胞EBV 感染模式;通过 KMH2-EBV 细胞与 HONE1、CNE1、CNE2、HK1 四株鼻咽癌细胞株分别直接接触培养,用Realtime-PCR检测各个细胞株的感染率,筛选感染率最高的细胞株构建鼻咽癌细胞EBV感染模型。然后采用RNA-Seq测序技术检测、比较分析共培养前、后HONE1 3d、5d、7d的细胞表达差异基因,运用生物信息学技术对测序所得结果进行分析。采用Realtime-PCR验证转录组测序数据候选基因。结果:1.通过携带EBV-GFP的KMH2与鼻咽癌细胞接触共培养方式,成功构建EBV感染的鼻咽癌细胞模型,其共培养条件下EBV感染效率明显高于EBV颗粒直接感染模式;2.转录组测序数据分析结果表明共培养前、后HONE1细胞的基因表达存在显著差异;3.GO功能显著富集的差异基因在EBV最初感染阶段主要涉及细胞免疫反应、抗原递呈、脂筏转运、细胞粘附等。随着感染推移,GO差异基因主要集中于于囊泡介导的转运、细胞内转运、代谢过程、大分子修饰等方面,而参与抗原递呈、免疫反应、凋亡过程、细胞死亡的基因大多数下调;4.KEGG数据库对差异表达基因Pathway功能富集分析主要集中于Phagosome、Focal adhesion、Herpes simplex infection、PI3K-Akt signaling pathway、Endocytosis等;5.在突变类型分析中,HONE1、HONE1 3d、HONE1 5d、HONE1 7d分别共获得12066、12185、11916、12267个SNP位点和2407、2678、2361、2695个INDEL位点,且突变类型以颠换为主,较多的突变类型发生在3'-UTR区。综合考虑共培养前后突变差异位点,共获得630个SNP位点及115个INDEL位点;6.Realtime-PCR方法验证2个差异表达基因,其差异表达倍数关系与表达谱测序结果总体趋势相一致,且在EBV感染的KMH2细胞及PBMC中,2个基因表达均上调,差异有统计学意义(P0.05)。结论:1.成功构建了鼻咽癌EBV感染细胞模型;2、建立体外EBV感染鼻咽癌细胞的差异基因表达谱,发现EBV感染前后的宿主细胞基因表达模式存在明显差异;3.测序结果表明EBV感染鼻咽癌细胞是一个多基因参与,多条通路涉及、病毒与宿主基因相互作用的过程。KEGG数据库对差异表达基因进行Pathway功能富集分析,推测EBV主要通过粘附、内吞等途径进入细胞。
[Abstract]:Objective: to screen and establish a suitable EBV infected nasopharyngeal carcinoma cell line model by direct infection of EBV particles and co-culture with EBV carrying Hodgkin's lymphoma cell line KMH2. The model was used to detect and analyze the differentially expressed genes in the time series of nasopharyngeal carcinoma (NPC) cells before and after infection with EBV in vitro using transcriptome sequencing technique, which laid a foundation for further exploring the molecular mechanism of EBV infection in epithelial cells. Methods: EBV-GFP, was prepared from AGS-EBV-GFP cells and EBV positive KMH2 cell lines were established. The EBV positive KMH2 cells were directly infected with the prepared EBV particles and cocultured directly by KMH2 cells carrying EBV with nasopharyngeal carcinoma cells. Screening suitable EBV infection model for nasopharyngeal carcinoma cells; KMH2-EBV cells were cultured directly with four HONE1,CNE1,CNE2,HK1 nasopharyngeal carcinoma cell lines. Realtime-PCR was used to detect the infection rate of each cell line and to screen the cell line with the highest infection rate to construct the EBV infection model of nasopharyngeal carcinoma cells. Then RNA-Seq sequencing technique was used to compare and analyze the differentially expressed genes in the cells before and after HONE1 3 days and 5 days after co-culture. The results of sequencing were analyzed by bioinformatics. Realtime-PCR was used to verify the candidate genes of transcriptome sequencing data. The result is 1: 1. The model of EBV infected nasopharyngeal carcinoma cells was successfully constructed by contact co-culture of KMH2 carrying EBV-GFP with nasopharyngeal carcinoma cells. The efficiency of EBV infection in co-culture was significantly higher than that of direct infection of EBV particles. 2. The results of transcriptome sequencing showed that there were significant differences in gene expression of HONE1 cells before and after co-culture. The differentially enriched genes of 3.GO are mainly involved in cellular immune response, antigen presentation, lipid raft transport, cell adhesion and so on in the initial stage of EBV infection. With the passage of infection, the differential genes of GO mainly focus on vesicular mediated transport, intracellular transport, metabolic process, macromolecular modification and so on, while most of the genes involved in antigen presentation, immune reaction, apoptosis and cell death are down-regulated. Pathway functional enrichment analysis of differentially expressed genes in 4.KEGG database was mainly focused on Phagosome,Focal adhesion,Herpes simplex infection,PI3K-Akt signaling pathway,Endocytosis. In the analysis of mutation types, 1 066 / 121855 SNP sites and 2407 INDEL loci were obtained respectively on day 17 of HONE1,HONE1 3d1 5d HONE15, and the mutation types were mainly transversion, and more mutation types occurred in 3'-UTR region. The mutation patterns were as follows: 1 067 SNP loci, 12267 SNP loci, and 2 697 INDEL loci, respectively, and most of the mutations occurred in the 3'-UTR region, and most of them were located in the 3'-UTR region. A total of 630 SNP loci and 115 INDEL loci were obtained considering the mutation differences before and after co-culture. Two differentially expressed genes were verified by 6.Realtime-PCR method. The relationship between the differential expression multiple and the result of expression profile sequencing was consistent with the general trend, and the two genes were up-regulated in KMH2 cells and PBMC infected with EBV. The difference was statistically significant (P0.05). Conclusion: 1. The cell model of nasopharyngeal carcinoma (NPC) infected with EBV was successfully constructed. (2) the differentially expressed gene profiles of nasopharyngeal carcinoma cells infected with EBV in vitro were established and the expression patterns of host genes were found to be significantly different before and after EBV infection. 3. Sequencing results showed that EBV infection in nasopharyngeal carcinoma cells was a multigene involved in the process of virus interaction with host genes. KEGG database was used to analyze the Pathway function enrichment of differentially expressed genes. Endocytosis and other pathways enter the cell.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.63
[Abstract]:Objective: to screen and establish a suitable EBV infected nasopharyngeal carcinoma cell line model by direct infection of EBV particles and co-culture with EBV carrying Hodgkin's lymphoma cell line KMH2. The model was used to detect and analyze the differentially expressed genes in the time series of nasopharyngeal carcinoma (NPC) cells before and after infection with EBV in vitro using transcriptome sequencing technique, which laid a foundation for further exploring the molecular mechanism of EBV infection in epithelial cells. Methods: EBV-GFP, was prepared from AGS-EBV-GFP cells and EBV positive KMH2 cell lines were established. The EBV positive KMH2 cells were directly infected with the prepared EBV particles and cocultured directly by KMH2 cells carrying EBV with nasopharyngeal carcinoma cells. Screening suitable EBV infection model for nasopharyngeal carcinoma cells; KMH2-EBV cells were cultured directly with four HONE1,CNE1,CNE2,HK1 nasopharyngeal carcinoma cell lines. Realtime-PCR was used to detect the infection rate of each cell line and to screen the cell line with the highest infection rate to construct the EBV infection model of nasopharyngeal carcinoma cells. Then RNA-Seq sequencing technique was used to compare and analyze the differentially expressed genes in the cells before and after HONE1 3 days and 5 days after co-culture. The results of sequencing were analyzed by bioinformatics. Realtime-PCR was used to verify the candidate genes of transcriptome sequencing data. The result is 1: 1. The model of EBV infected nasopharyngeal carcinoma cells was successfully constructed by contact co-culture of KMH2 carrying EBV-GFP with nasopharyngeal carcinoma cells. The efficiency of EBV infection in co-culture was significantly higher than that of direct infection of EBV particles. 2. The results of transcriptome sequencing showed that there were significant differences in gene expression of HONE1 cells before and after co-culture. The differentially enriched genes of 3.GO are mainly involved in cellular immune response, antigen presentation, lipid raft transport, cell adhesion and so on in the initial stage of EBV infection. With the passage of infection, the differential genes of GO mainly focus on vesicular mediated transport, intracellular transport, metabolic process, macromolecular modification and so on, while most of the genes involved in antigen presentation, immune reaction, apoptosis and cell death are down-regulated. Pathway functional enrichment analysis of differentially expressed genes in 4.KEGG database was mainly focused on Phagosome,Focal adhesion,Herpes simplex infection,PI3K-Akt signaling pathway,Endocytosis. In the analysis of mutation types, 1 066 / 121855 SNP sites and 2407 INDEL loci were obtained respectively on day 17 of HONE1,HONE1 3d1 5d HONE15, and the mutation types were mainly transversion, and more mutation types occurred in 3'-UTR region. The mutation patterns were as follows: 1 067 SNP loci, 12267 SNP loci, and 2 697 INDEL loci, respectively, and most of the mutations occurred in the 3'-UTR region, and most of them were located in the 3'-UTR region. A total of 630 SNP loci and 115 INDEL loci were obtained considering the mutation differences before and after co-culture. Two differentially expressed genes were verified by 6.Realtime-PCR method. The relationship between the differential expression multiple and the result of expression profile sequencing was consistent with the general trend, and the two genes were up-regulated in KMH2 cells and PBMC infected with EBV. The difference was statistically significant (P0.05). Conclusion: 1. The cell model of nasopharyngeal carcinoma (NPC) infected with EBV was successfully constructed. (2) the differentially expressed gene profiles of nasopharyngeal carcinoma cells infected with EBV in vitro were established and the expression patterns of host genes were found to be significantly different before and after EBV infection. 3. Sequencing results showed that EBV infection in nasopharyngeal carcinoma cells was a multigene involved in the process of virus interaction with host genes. KEGG database was used to analyze the Pathway function enrichment of differentially expressed genes. Endocytosis and other pathways enter the cell.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.63
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相关期刊论文 前10条
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