Establishment of a new low-density cDNA macroarray and the application in the activity of IFN against HBV / 中华实验和临床病毒学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expression profile of genes which are involved in IFN antiviral activity and IFN signal transduction pathway in Hep G2 and HepG2.2.15 cells.</p><p><b>METHODS</b>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 cDNA inserts were amplified by PCR and then were spotted onto the Hybond-N+ membranes. The membranes were denatured and neutralized for Macroarray analysis. HepG2.2.15 and Hep G2 cells were treated without or with IFN-alpha for 6 h, and the total cellular RNA was isolated using Trizol Reagent. Radio-labelled cDNA was generated from 20 microgram of RNA by reverse transcription using 360 units of reverse transcriptase in the presence of 30 microCi of alpha-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 OptiQuant Imager Analysis Software and converted into digital data.</p><p><b>RESULTS</b>The authors found that just partially IFN-inducible genes were expressed in Hep G2 and HepG2.2.15 cells, and the majority of IFN-inducible genes was lowly responsive or non-responsive to IFN-a treatment. Some interferon-stimulated genes (ISGs) were inhibited or blocked, especially in HepG2.2.15 cells. Interestingly, the authors found that the IFN signal transduction pathway (Jak-STAT) was intact and unimpaired in HepG2.2.15 cells.</p><p><b>CONCLUSION</b>Differential gene expression profiles in response to IFN were found between Hep G2 and HepG2.2.15 cells.</p>