ABSTRACT
AIMS/HYPOTHESIS: The regenerative process in the pancreas is of particular interest, since insulin-producing beta cells are lost in diabetes. Differentiation of new beta cells from pancreatic non-endocrine cells has been reported in vivo and in vitro, a finding that implies the existence of pancreatic stem/progenitor cells. However, while tissue-specific stem cells are well documented in skin, intestine and testis, pancreatic stem cells have been elusive. We hypothesised that pancreatic stem/progenitor cells within the non-endocrine fraction could be a source of new islets in vitro. METHODS: To test if there were such cells within the pancreas, we generated pancreatic cell aggregates from tissue remaining after islet isolation from mouse insulin promoter 1-green fluorescent protein (MIP-GFP) mice. To eliminate any contamination of insulin-positive cells, we deleted all GFP-positive aggregates using COPAS Select and cultured with Matrigel. Immunohistochemistry, quantitative real-time PCR and single-cell nested RT-PCR were performed to confirm formation of insulin-producing cells. RESULTS: The GFP-negative cells were expanded as monolayers and then differentiated into three-dimensional cystic structures. After 1 week of culture, GFP-positive cells were found as clusters or single cells. By quantitative real-time PCR, no insulin mRNA was detected immediately after COPAS sorting, but after differentiation insulin mRNA of the whole preparation was 1.91 +/- 0.31% that of purified MIP-GFP beta cells. All GFP-positive cells expressed insulin 1; most expressed insulin 2, pancreas duodenum homeobox-1 and cytokeratin 19 by single cell nested RT-PCR. CONCLUSIONS/INTERPRETATION: Our data support the concept that within the exocrine (acinar and ductal) pancreas of the adult mouse there are cells that can give rise to insulin-positive cells in vitro.
Subject(s)
Cell Separation/methods , Insulin-Secreting Cells/cytology , Islets of Langerhans/cytology , Animals , Cell Culture Techniques , Diabetes Mellitus, Type 1/surgery , Flow Cytometry , Genes, Reporter , Green Fluorescent Proteins/genetics , Insulin/genetics , Insulin-Secreting Cells/physiology , Insulin-Secreting Cells/transplantation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pancreas/cytology , Pancreas/physiology , Pancreatic Ducts/cytology , Promoter Regions, Genetic , RNA, Messenger/analysis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
It has not been clarified yet as to whether hypoxia and inflammation affect NO synthesis. In this study, we investigated the transcription of inducible nitric oxide synthase (iNOS) mRNA and the production of nitric oxide (NO) in rat smooth muscle cells (SMCs) cultured under hypoxic conditions in the presence and absence of proinflammatory cytokine interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). We found that hypoxia inhibited the production of NO but did not affect the transcription of iNOS mRNA in rat SMCs treated with IFN-gamma, LPS, or both. These results indicate that O(2) is involved in the regulation of NO synthesis in inflammatory tissues.
Subject(s)
Muscle, Smooth, Vascular/metabolism , Nitric Oxide Synthase/genetics , Nitric Oxide/metabolism , Oxygen/metabolism , Animals , Cell Hypoxia/physiology , Cells, Cultured , Drug Interactions , Gene Expression , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Male , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/enzymology , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , RNA, Messenger/metabolism , Rats , Rats, Wistar , Time Factors , Transcription, Genetic/drug effectsABSTRACT
BACKGROUND: Studies in corneal transplant rejection remain important because acute immunologic rejection continues to be the leading cause of human corneal transplant failure. As the permeability of vessels and the neovascularization induce cells infiltration into the graft, we considered the possibility that vascular endothelial growth factor (VEGF), a potent permeability-increasing factor and angiogenesis-mediating factor, could participate in the immune response. METHODS: As the established corneal transplant model for rejection, the corneal transplant between Lewis and Fisher rats has been reported. First, we evaluated VEGF production in the graft by immunohistochemical method in the animal model. Next, we tried to neutralize the effect of VEGF by topical administration of anti-VEGF antibody. We administered anti-VEGF antibody as eye drops for 10 days just after the transplantation of the established animal corneal transplant model. RESULTS: VEGF was strongly produced from the infiltrative cells into the graft. Anti-VEGF antibody significantly suppressed the acute rejection compared with saline or rabbit IgG. CONCLUSIONS: The inhibition of VEGF by topically applied neutralizing antibody is a new potential therapeutic strategy for the treatment of corneal transplantation.
