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

发布时间：2018-07-03 17:14

本文选题：丙型肝炎病毒 + 非结构蛋白B　；参考：《中南大学学报(医学版)》2014年02期

【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、细胞周期和细胞增殖等有关的多种基因表达,主要影响与细胞凋亡、细胞周期和癌症相关的信号转导通路。
[Abstract]:Objective: to screen the differential expression gene and gene pathway of hepatitis C virus (HCV) 1b non structural protein 4B (nonstructural protein 4B, NS4B) in the lentivirus stable cell line, and to provide the basis for the in-depth study of the role and mechanism of HCV gland in the development of chronic hepatitis C and hepatocellular carcinoma. The 2 lentivirus stable cell lines LO2-NS4B and the negative control lentivirus stable cell line LO2-mkate2 were recovered and amplified, and the differential expression genes of LO2-NS4B and LO2-mkate2 were screened by Human Gene 1.0ST chip. Real-time quantitative polymerase chain reaction (real-time QPCR) was used to verify the signal transduction pathway of differential genes, and 5 genes in the gene chip, namely, protein kinase C delta binding protein (protein), were tested by real time quantitative polymerase chain reaction (polymerase chain reaction, real-time QPCR). Kinase C delta binding protein, PRKCDBP) gene, the tumor protein p53 (tumor protein p53, TP53) gene, and the oncogene homologous gene of the murine thymoma virus 1, containing 3 baculovirus apoptosis protein inhibitory factor repeat sequences MRNA levels of the 2 B-cell lymphoma 2-like1 (BCL2L1) gene for B cell lymphoma. Results: the ratio of the gene fluorescence intensity between LO2-NS4B and LO2-mkate2 is greater than 1.2 or less than 0.8, and there are 2682 differentially expressed genes in L02-NS4B among the 28869 known human genes, including 1446 gene expression up-regulated and 1. 236 genes were down regulated. There were 41 significant signal transduction pathways involved in the regulation of gene involvement, including apoptosis pathway, extracellular matrix receptor interaction pathway, cell cycle pathway, cancer pathway and Toll like receptor signaling pathway, and down regulated genes involved in significant signal transduction pathways, including cancer pathway, Wnt signaling pathway, and 20 signaling pathways. The cell cycle pathway, such as.Real-time QPCR, confirmed that 3 gene expression changes in 5 up-regulated genes were consistent with gene chip results, which were AKT1, BIRC3 and BCL2L1, and the anastomosis rate was 60%. conclusion: HCV NS4B can regulate a variety of gene expression related to apoptosis, cell cycle and cell proliferation in LO2 cells, which mainly affect the cells and cells. Apoptosis, cell cycle and cancer related signal transduction pathways.
【作者单位】：中南大学湘雅医院感染科;南华大学附属第一医院感染科;南华大学流行病学教研室;
【分类号】：R373.21

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