Purchase this article with an account.
GS Schultz, TD Blalock, JC Varela, HV Baker, GR Grotendorst; Effect of Connective Tissue Growth Factor on Gene Expression Patterns in Human Corneal Fibroblast Cultures . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4010.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the effects of connective tissue growth factor (CTGF) on gene expression in cultures of human corneal fibroblasts. Methods: Human corneal fibroblasts were grown to confluence in ten 75 cm2 tissue culture flasks in serum-supplemented medium, then the cells were shifted to serum free medium for 48 hours. After serum starvation, the cells were exposed to recombinant human CTGF at 25 ng/mL. At five time points after exposure to CTGF (0, 4, 8, 16, and 24 hours), total RNA was isolated from duplicate cultures of fibroblasts using Qiagen RNeasy kit, mRNA was reverse transcribed using a T7 RNA polymerase-tagged primer, and cDNA was transcribed to labeled cRNA with biotinylated nucleotides. The labeled cRNA was fragmented and hybridized to Affymetrix Test 3 and U95a human chips, which contain 9,170 known genes and 3,389 expressed sequenced tags (EST). Fluorescent intensities were measured, globally normalized, filtered, and the data analyzed with Cluster and TreeView programs to reveal genes that have similar patterns of expression. Results: A total of 5,335 known genes and 2,387 ESTs were grouped into 10 different clusters based on the similarities of their gene expression patterns. Many wound healing-related genes were significantly altered by CTGF. Numerous genes were significantly up regulated such as VEGF, TIMPs, IL-1b, IL-15, IL-8, HSPG, and several types of collagen, while other genes were significantly down regulated such as IL-1-R, IL-7-R, cadherin, IGFBP5, IGFBP2, and TRAP1. Conclusion: CTGF regulates expression of a large number of genes in cultured human corneal fibroblasts. Hierarchical clustering analysis of gene expression data at 5 time points up to 24 hours after exposure to CTGF identified 10 cluster groups, and included genes that are rapidly induced by CTGF while some genes were rapidly down regulated. Hierarchical clustering of genes based on changes in levels of gene expression determined by microarray analysis identified a rather small number of clusters of genes and suggests the possibility of those genes having common regulatory pathways.
This PDF is available to Subscribers Only