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Gene Expression in an In Vitro Model of Epithelial Repair* FREE TO VIEW

John Spurzem, MD; Ji Zhang PhD; Xiang-der Liu, MD; Stephan I. Rennard, MD; Debra V. Romberger, MD
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*From the Omaha Veterans Affairs Medical Center and University of Nebraska Medical Center, Omaha, NE.

Correspondence to: John R. Spurzem, MD, Associate Professor of Medicine, Pulmonary and Critical Care Medicine, University of Nebraska Medical Center, 985300 Nebraska Medical Center, Omaha, NE 68198-5300

Chest. 2002;121(3_suppl):79S-80S. doi:10.1378/chest.121.3_suppl.79S
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Epithelial repair is likely very important in the pathogenesis of respiratory disorders. Disordered repair is important in the pathogenesis of COPD. Epithelial repair requires cells to alter their interactions with extracellular matrix such that they can attach to and migrate over the inflammatory, provisional matrix. We hypothesize that epithelial cell migration after injury is under the control of distinct gene clusters associated with cell proliferation, tissue remodeling, and re-epithelialization. In order to address this hypothesis, we have utilized an in vitro model of wounding an epithelial cell monolayer, quantitative polymerase chin reaction, and microarray gene expression analysis. A transformed, but nontumorigenic, bronchial epithelial cell line, BEAS-2B cells, was grown into confluent monolayers that were then wounded with a specialized cell rake. At sequential time points, the cells were harvested for RNA extraction and analysis by quantitative polymerase chain reaction (Taqman process) [model 7700 sequence detector; Applied Biosystems; Foster City, CA]. We first evaluated the time course of expression of four genes that we believed would be relevant to repair and migration (fibronectin, transforming growth factor-β2, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1). Expression of glyceraldehyde-3-phosphate dehydrogenase was used for normalization. The expression of all the genes was increased with peaks at 12 h after wounding. The experiment was repeated with primary cultures of normal human bronchial epithelial cells with similar results. The increases in expression were more than fourfold in all cases, demonstrating that the in vitro wounding model created detectable changes in relevant gene expression. We next evaluated gene expression using cell monolayers 12 h after wounding with microarray gene expression analysis. We used a human complementary DNA library (ResGen; Huntsville, AL) arrayed onto glass slides (GMS417 Arrayer; Affymetrix; Santa Clara, CA). Analysis with a GenePix 4000 scanner (Axon Instruments; Union City, CA) and its software package, GenePix Pro 3.0, demonstrated 459 genes that had a more than threefold increase in expression over control expression. This group of genes included the integrin-β1 chain, collagen-related genes, and fibronectin. In vitro wounding of cell monolayers induces detectable increases in relevant gene expression that should allow study of groups of genes involved in epithelial repair.




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