Isolation of genes involved in pancreas regeneration by subtractive hybridization

Abstract

The deterioration of β cells in the pancreas is a crucial factor in the progression of diabetes mellitus; therefore, the recovery of β cells is of vital importance for effective diabetic therapeutic strategies. Partially pancreatectomized rats have been used for the investigation of pancreatic regeneration. Because it was determined that tissue extract from the partially-dissected pancreas induces pancreatic differentiation in embryonic stem cells, paracrine factors were thought to be involved in the regeneration. In this study, we screened for genes that had higher mRNA levels 2 days after 60%-pancreatectomy. The genes were isolated using subtractive hybridization and DNA sequencing. Twelve genes (adipsin, Aplp2, Clu, Col1a2, Glul, Krt8, Lgmn, LOC299907, LOC502894, Pla2g1b, Reg3α and Xbp1) were identified, and RT-PCR and real-time PCR analyses were performed to validate their expression levels. Among the genes identified, three genes (Glul, Lgmn and Reg3a) were selected for further analyses. Assays revealed that Glul and Reg3α enhance cell growth. Glul, Lgmn and Reg3α change the expression level of islet marker genes, where NEUROD, NKX2.2, PAX4 and PAX6 are up-regulated and somatostatin is down-regulated. Thus, we believe that Glul, Lgmn and Reg3a can serve as novel targets in diabetes mellitus genetic therapy.