筛选丙型肝炎病毒1b型非结构蛋白4B慢病毒L02稳定株差异表达基因和基因通路  

Screening of differentially expressed genes and gene pathways in hepatitis C virus 1b type nonstructural protein 4B stably expressed L02 cell line

作　　者：蒋孝华[1,2] 谢玉桃[1] 蔡亚平[3] 雷创[2] 江波[2] 彭花[2] 

机构地区：[1]中南大学湘雅医院感染科,长沙410087 [2]南华大学附属第一医院感染科,湖南衡阳421001 [3]南华大学流行病学教研室,湖南衡阳421001

出　　处：《中南大学学报（医学版）》2014年第2期117-123,共7页Journal of Central South University ：Medical Science

摘　　要：目的:筛选表达丙型肝炎病毒(hepatitis C virus,HCV)1b型非结构蛋白4B(nonstructural protein 4B,NS4B)的慢病毒稳定细胞株L02-NS4B差异表达基因和基因通路,为深入研究HCV NS4B在慢性丙型肝炎和肝细胞癌发生中的作用及机制提供依据。方法:将已构建好的2株慢病毒稳定细胞株LO2-NS4B和阴性对照慢病毒稳定细胞株LO2-mkate2复苏扩增;应用Human Gene 1.0ST芯片筛选出LO2-NS4B与LO2-mkate2差异表达基因。基于京都基因和基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)数据库,利用Fisher精确检验和卡方检验,对差异基因参与的信号转导通路进行显著性分析。应用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,real-time QPCR)方法验证基因芯片中5个表达上调且与凋亡有关的基因,即蛋白激酶Cδ结合蛋白(protein kinase C delta binding protein,PRKCDBP)基因、肿瘤蛋白p53(tumor protein p53,TP53)基因、v-akt鼠科胸腺瘤病毒癌基因同源物1(v-akt murine thymoma viral oncogene homolog 1,AKT1)基因、含3个杆状病毒凋亡蛋白抑制因子重复序列(baculoviral IAP repeat containing 3,BIRC3)基因和B细胞淋巴瘤2样1基因(B-cell lymphoma 2-like1,BCL2L1)的mRNA水平。结果:以LO2-NS4B与LO2-mkate2之间基因荧光强度的比值大于1.2或小于0.8为差异表达基因,在已知的28 869个人类基因中L02-NS4B中有2 682个差异表达基因,包括1 446个基因表达上调和1 236个基因表达下调。上调基因参与的显著性信号转导通路41项,主要有凋亡通路、细胞外基质受体相互作用通路、细胞周期通路、癌症通路和Toll样受体信号通路等;下调基因参与的显著性信号转导通路20项,主要有癌症通路、Wnt信号通路和细胞周期通路等。Real-time QPCR验证5个表达上调基因中有3个基因表达变化与基因芯片结果一致,分别是AKT1,BIRC3和BCL2L1,吻合率为60%。结论:HCV NS4B可以调节LO2细胞中与细胞凋亡、细胞周Objective: To screen differentially expressed genes and gene pathways in L02 cell line stably expressing hepatitis C virus(HCV) Ib type nonstructural protein 4B(NS4B) mediated by lentiviral system, and to provide a basis for further research of molecular biological mechanism of NS4B gene in chronic hepatitis C and hepatocarcinogenesis. Methods: NS4B stably overexpressed L02 cell line and negative control stable L02 cell line, designated as L02-NS4B and L02-mkate2 respectively, were resurrected and amplified in vitro. The differentially expressed genes between L02-NS4B and L02-mkate2 were determined by gene expression microarray from Human Gene 1.0ST. h e signii cant pathways of the dif erential genes were selected by the Fisher's exact test and χ2 test according to kyoto encyclopedia of genes and genomes(KEGG) database. The differential expression levels of 5 selected genes including protein kinase C delta binding protein(PRKCDBP), tumor protein p53(TP53), v-akt murine thymoma viral oncogene homolog 1(AKT1), baculoviral IAP repeat containing 3(BIRC3) and B-cell lymphoma 2-like1(BCL2L1) from cDNA microarray data were further verii ed by real-time quantitative polymerase chain reaction(real-time QPCR). Results: Between L02-NS4B and L02-mkate2, the genes with l urescence intensity ratio >1.2 or <0.8 were considered as differentially expressed genes. A total of 2 682 differentially expressed genes in the known 28 869 human genes were detected in L02-NS4B, 1 446 genes were upregulated and 1 236 genes were downregulated. A total of 41 involved pathways of up-regulated dif erential genes were identii ed by KEGG database, mainly including apoptosis, extracellular matrix receptor interaction, cell cycle, pathways in cancer and Toll-like receptor signaling pathway; and 20 involved pathways of down-regulated dif erential genes were identii ed, mainly including pathways in cancer, Wnt signaling pathway and cell cycle pathway. Of the 5 upregulated genes selected from cDNA microarray data, 3 genes showed the same dif erential

关 键 词：丙型肝炎病毒 非结构蛋白4B 基因芯片 慢病毒 

分 类 号：R373.21[医药卫生—病原生物学]