Isolation of genes involved in pancreas regeneration by subtractive hybridization.
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.
Choi
Jong-Ho
JH
College of Medicine, CHA University, CHA General Hospital, Seoul 135-081, Korea.
Lee
Min-Young
MY
Kim
Yoolee
Y
Shim
Jeong-Yun
JY
Han
Sang-Moon
SM
Lee
Kyung-Ah
KA
Choi
Young-Kil
YK
Jeon
Hae-Myung
HM
Baek
Kwang-Hyun
KH
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016716:PC12 Cells; D010179:Pancreas / Q000378:metabolism*; D000073718:Pancreatitis-Associated Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D012038:Regeneration / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors; D000099240:Asparaginyl Endopeptidase
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D003546:Cysteine Endopeptidases; D018076:DNA, Complementary; D005974:Glutamate-Ammonia Ligase; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D037181:Lectins, C-Type; D009687:Nuclear Proteins; D000073718:Pancreatitis-Associated Proteins; D014157:Transcription Factors; D000099240:Asparaginyl Endopeptidase; C000715631:NKX2-2 protein, human; C000715628:Nkx2-2 protein, rat; C000612141:REG3A protein, human; C000612146:Reg3a protein, rat