Differentially up-regulated genes in proliferating porcine neonatal pancreas cells caused by epidermal growth factor

Pancreatic duct cells are considered to be a major source for beta-cell regeneration or neogenesis. Although epidermal growth factor (EGF) is a well-known important growth factor for pancreas development, the control of pancreatic duct cell growth and differentiation by EGF is poorly understood. In this study, we focused on identifying the genes that were differentially up-regulated in response to EGF stimulation using monolayer cultured porcine neonatal pancreas cells. Cells were obtained from 1 to 3 day old pigs, dispersed and cultured for 8 days. Monolayer cultured porcine pancreas cells were comprised of duct cells and some endocrine and mesenchymal cells (75.2+/-15.1, 19.6+/-4.9, and 9.5+/-3.1%, respectively). After 16 h in serum free media, cells were treated with 100 mug/L EGF for 24 h. Differentially expressed genes were screened by subtractive hybridization. H-3-thymidine uptake was significantly increased by EGF with time (untreated vs. 24 h treated, untreated vs. 48 h treated: 305.5+/-3.5 cpm vs. 380.3+/-17.3 cpm (P<0.05), 309.2 +/- 4.51 vs. 929 +/- 9.19 cpm, (P<0.005), respectively). Three hundred and fify cDNA clones were obtained by subtractive hybridization and the inserts were confirmed in 161 colonies and then sequenced. Finally, we found increased mRNA expression of five unknown and five known genes, including cytochrome c oxidase subunit I (COI), cyclooxygenase-2 (COX-2), matrix metalloproteinase-13 (MMP-13), Wiskott-Aldrich syndrome protein interacting protein (WASPIP), and hyaluronan synthase-2 (HAS-2). We confirmed the up-regulation of these genes by Northern blot and semi-quantitative RT-PCR at various time points. The present findings opened new targets for the research on the mechanisms of pancreatic duct cell proliferation by EGF.