鼻咽癌上皮細(xì)胞EB病毒感染模型的構(gòu)建及其相關(guān)基因的篩選
發(fā)布時(shí)間：2018-11-18 15:55
【摘要】：目的:通過(guò)EBV顆粒直接感染以及與攜帶EBV的霍奇金淋巴瘤細(xì)胞KMH2共培養(yǎng)兩種方式,篩選并建立適宜的EBV感染鼻咽癌細(xì)胞系模型。進(jìn)而通過(guò)該模型利用轉(zhuǎn)錄組測(cè)序技術(shù)檢測(cè)分析鼻咽癌細(xì)胞體外感染EBV前后時(shí)間序列的差異表達(dá)基因,為進(jìn)一步探討EBV感染上皮細(xì)胞的分子機(jī)制奠定基礎(chǔ)。方法:通過(guò)AGS-EBV-GFP細(xì)胞制備EBV-GFP,并建立EBV陽(yáng)性的KMH2細(xì)胞株,用制備的EBV顆粒直接感染鼻咽癌細(xì)胞及用攜帶EBV的KMH2細(xì)胞與鼻咽癌細(xì)胞直接接觸共培養(yǎng)方式,篩選適宜的鼻咽癌細(xì)胞EBV 感染模式;通過(guò) KMH2-EBV 細(xì)胞與 HONE1、CNE1、CNE2、HK1 四株鼻咽癌細(xì)胞株分別直接接觸培養(yǎng),用Realtime-PCR檢測(cè)各個(gè)細(xì)胞株的感染率,篩選感染率最高的細(xì)胞株構(gòu)建鼻咽癌細(xì)胞EBV感染模型。然后采用RNA-Seq測(cè)序技術(shù)檢測(cè)、比較分析共培養(yǎng)前、后HONE1 3d、5d、7d的細(xì)胞表達(dá)差異基因,運(yùn)用生物信息學(xué)技術(shù)對(duì)測(cè)序所得結(jié)果進(jìn)行分析。采用Realtime-PCR驗(yàn)證轉(zhuǎn)錄組測(cè)序數(shù)據(jù)候選基因。結(jié)果:1.通過(guò)攜帶EBV-GFP的KMH2與鼻咽癌細(xì)胞接觸共培養(yǎng)方式,成功構(gòu)建EBV感染的鼻咽癌細(xì)胞模型,其共培養(yǎng)條件下EBV感染效率明顯高于EBV顆粒直接感染模式;2.轉(zhuǎn)錄組測(cè)序數(shù)據(jù)分析結(jié)果表明共培養(yǎng)前、后HONE1細(xì)胞的基因表達(dá)存在顯著差異;3.GO功能顯著富集的差異基因在EBV最初感染階段主要涉及細(xì)胞免疫反應(yīng)、抗原遞呈、脂筏轉(zhuǎn)運(yùn)、細(xì)胞粘附等。隨著感染推移,GO差異基因主要集中于于囊泡介導(dǎo)的轉(zhuǎn)運(yùn)、細(xì)胞內(nèi)轉(zhuǎn)運(yùn)、代謝過(guò)程、大分子修飾等方面,而參與抗原遞呈、免疫反應(yīng)、凋亡過(guò)程、細(xì)胞死亡的基因大多數(shù)下調(diào);4.KEGG數(shù)據(jù)庫(kù)對(duì)差異表達(dá)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.在突變類型分析中,HONE1、HONE1 3d、HONE1 5d、HONE1 7d分別共獲得12066、12185、11916、12267個(gè)SNP位點(diǎn)和2407、2678、2361、2695個(gè)INDEL位點(diǎn),且突變類型以顛換為主,較多的突變類型發(fā)生在3'-UTR區(qū)。綜合考慮共培養(yǎng)前后突變差異位點(diǎn),共獲得630個(gè)SNP位點(diǎn)及115個(gè)INDEL位點(diǎn);6.Realtime-PCR方法驗(yàn)證2個(gè)差異表達(dá)基因,其差異表達(dá)倍數(shù)關(guān)系與表達(dá)譜測(cè)序結(jié)果總體趨勢(shì)相一致,且在EBV感染的KMH2細(xì)胞及PBMC中,2個(gè)基因表達(dá)均上調(diào),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.成功構(gòu)建了鼻咽癌EBV感染細(xì)胞模型;2、建立體外EBV感染鼻咽癌細(xì)胞的差異基因表達(dá)譜,發(fā)現(xiàn)EBV感染前后的宿主細(xì)胞基因表達(dá)模式存在明顯差異;3.測(cè)序結(jié)果表明EBV感染鼻咽癌細(xì)胞是一個(gè)多基因參與,多條通路涉及、病毒與宿主基因相互作用的過(guò)程。KEGG數(shù)據(jù)庫(kù)對(duì)差異表達(dá)基因進(jìn)行Pathway功能富集分析,推測(cè)EBV主要通過(guò)粘附、內(nèi)吞等途徑進(jìn)入細(xì)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.63
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相關(guān)期刊論文 前10條
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