Analysis of globle gene expression changes observed in an in vitro fibroblast model of IPF

The activated fibroblast is the putative effector cell for the progressive fibrotic phenotype idiopathic pulmonary fibrosis (IPF). Recent studies investigating global gene expression differences between normal and IPF fibroblasts indicate that changes in gene expression occur in these fibroblasts in culture. Employing a technique that minimizes cellular phenotypic alterations, we characterized the global gene expression changes in pulmonary fibroblasts by comparing both cultured and non-cultured IPF and normal cells. The results revealed dramatic difference between IPF and normal fibroblast as they progressed in culture. While there are significant differences in gene expression between IPF and normal fibroblast at P0, after 3 passages in culture, no statistically significant difference was observed. This study is a comprehensive investigation of gene expression within IPF and normal fibroblasts in culture and sheds light on the efficacy of in vitro models of pulmonary fibroblasts. 24 total pulmonary fibroblast samples were used in this study. These fibroblasts were isolated from 8 lungs with end-stage idiopathic pulmonary fibrosis and 4 normal donors lungs (controls) designated brain dead, non-diseased but not suitable for transplantation. 16 IPF samples included 8 non-cultured (P0) and 8 cultured (P3). 8 normal samples included 4 non-cultured (P0) and 4 cultured (P3). RNA extraction was carried out using Qiagen RNeasy Kit. Experimental/control samples were amplified amino-allylated RNA labeled with Cy5 and Stratagene Reference RNA was amplified and amino-allylated and labeled with Cy3. One round of amplification was carried out using Ambion MessageAmp II kit with amino-allylated UTP according to the protocol of the Duke University Institute for Genome Sciences and Policy. Amplification and amino-allylation of the Stratagene Reference RNA and Hybridization of Reference with all samples were carried out by the Duke Institute for Genomic Sciences and Policy.