新型低密度cDNA Macroarray的建立及其应用于干扰素抗HBV的研究  

Establishment of a new low-density cDNA Macroarray and the application in the activity of IFN against HBV

作　　者：管世鹤[1] 刘华平[1] 杨东亮[2] 陆蒙吉 Michael Roggendorf Joerg F.Schlaak 

机构地区：[1]安徽医科大学第一附属医院检验科免疫室,合肥230022 [2]华中科技大学同济医学院附属同济医院临床免疫研究室 [3]德国Duisburg-Essen大学医学院病毒学研究所 [4]德国Duisburg-Essen大学医学院胃肠和肝脏病科

出　　处：《中华实验和临床病毒学杂志》2005年第3期236-239,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的了解人肝胚瘤细胞株HepG2和稳定转染HBV全基因组细胞株HepG2.2.15的干扰素(IFN)抗病毒基因表达谱。方法选择与IFN抗病毒及其信号转导途径相关的288个IMAGE克隆,经聚合酶链反应(PCR)扩增相应的基因片段,将扩增的DNA片段纯化后点样于阳离子尼龙膜上以制备成人工芯片。HepG2和HepG2.2.15细胞经IFN处理后,分离细胞总RNA,进行逆转录并且同时以32P标记;将标记的cDNA靶基因与尼龙膜上的探针进行分子杂交。经Cyclone扫描系统和OptiQuantTM软件处理,将图像信号转换成数据信息;再经Excel软件分析、处理形成Macroarray数据值。结果Macroarray分析提示,在HepG2和HepG2.2.15细胞仅有部分IFN诱导基因表达,多数IFN诱导基因呈低表达或不表达。通过比较HepG2和HepG2.2.15细胞,发现两者表达IFN诱导基因有差异,即对IFN应答有差异。进一步分析还发现,尽管与HepG2细胞存在差异的IFN应答,HepG2.2.15细胞的IFN信号转导途径(JakSTAT途径)却是开放的、未受损伤的。结论HepG2和HepG2.2.15细胞具有差异的IFN抗病毒基因表达谱。Objective To investigate the expression profile of genes which are involved in IFN anliviral activity and IFN signal transduetion pathway in Hep G2 and HepG2.2. 15 cells. Methods Genes of interest were selected from the UniGene database （http：//www. ncbi. nlm. gov/UniGene/Hs. Home. html） . The 5＇IMAGE clones With 0. 5-0. 8 kb length were chosen and ordered from RZPD company. The eDNA inserts were amplified by PCR and then were spotted onto the Hvbond-N ^＋ membranes. The membranes were denatured and neutralized for Maeroarray analysis. HepG2.2. 15 and Hep G2 cells were treated without or with IFN-α for 6 h, and the total cellular RNA was isolated using Trizol Reagent. Radio-labelled eDNA was generated from 20 μg of RNA by reverse transcription using 360 units of reverse transeriptase in the presence of 30 μCi of α-^32p dCTP. Hybridization was performed between ^32P-labelled cDNA and membrane arrays. The membranes were then scanned, and the intensity of autoradiographic spots was quantitated by Cyclone Storage Phosphor System. The images were subsequently analysed by the OptiQuantTM Imager Analysis Software and converted into digital data. Results The authors found that just partially IFN- inducible genes were expressed in Hep G2 and HepG2.2. 15 cells, and the majority of IFN-indueible genes was lowly responsive or non-responsive to IFN-α treatment. Some interferom-stimulated genes （ISGs） were inhibited or blocked, especially in HepG2.2. 15 cells. Interestingly, the authors found that the IFN signal transduetion pathway （Jak-STAT） was intact and unimpaired in HepG2.2. 15 cells. Conclusion Differential gene expression profiles in response to IFN were found between Hep G2 and HepG2.2. 15 cells.

关 键 词：肝炎病毒 乙型 干扰素类 CDNA Macroarray技术 表达的序列标记 MACROARRAY HEPG2.2.15细胞 抗HBV 干扰素 聚合酶链反应(PCR) 

分 类 号：R346[医药卫生—基础医学]