ABSTRACT
Investigation of pancreatic interstitial fibroblasts has proven difficult in situ. We have established a method for the isolation of pancreatic fibroblastoid/stellate cells by outgrowth from pancreatic tissue explanted into culture dishes. This technique gives a high yield of viable cells from small tissue samples. Outgrown fibroblastoid cells were established as a primary cell line and characterized during long-term culture. We investigated the development of stellate cell markers, i.e. fat storage, expression of desmin, and alpha-smooth muscle actin (alphaSMA), over weeks in culture. AlphaSMA, investigated by indirect immunofluorescence staining and Western blot analysis, revealed a constant rise in expression during routine culture. After 13 passages. approximately 100% of cells were positive for alphaSMA expression, indicating a myofibroblast type of differentiation in vitro.
Subject(s)
Cell Culture Techniques/methods , Fibroblasts/cytology , Pancreas/cytology , Actins/analysis , Animals , Azo Compounds , Biomarkers , Blotting, Western , Cell Differentiation , Cell Line , Cell Separation , Ceruletide/toxicity , Coloring Agents , Contact Inhibition , Culture Media , Desmin/analysis , Fluorescent Antibody Technique, Indirect , Lipids/analysis , Male , Muscle, Smooth/chemistry , Muscle, Smooth/ultrastructure , Pancreas/drug effects , Pancreatitis/chemically induced , Pancreatitis/metabolism , Pancreatitis/pathology , Rats , Rats, Wistar , Staining and LabelingABSTRACT
TGFbeta1 is a multifunctional factor, controlling cellular growth and extracellular matrix production. Deletion of the TGFbeta1 gene in mice results in multiple inflammatory reactions. Targeted overexpression of TGFbeta1 in pancreatic islet cells leads to fibrosis of the exocrine pancreas in transgenic mice. In pancreatic fibrosis interstitial fibroblasts are primary candidates for production and deposition of extracellular matrix. Still, little is known about regulation of these cells during development of pancreatic disease. We established primary cell lines of pancreatic fibroblastoid/stellate cells (PFC) from rat pancreas. Investigation of rPFCs in vitro shows TGFbeta1 expression by RT-PCR analysis. Mature TGFbeta1 was detected in culture supernatants by immunoassay. Rat PFCs in culture possess both receptors TGFbeta receptor type I, and type II, necessary for TGFbeta1 signal transduction. Inhibition of TGFbeta1 activity by means of neutralizing antibodies interferes with an autocrine loop and results in a 2-fold stimulation of cell growth. So far, pancreatic fibroblastoid/stellate cells in vitro were known as a target of TGFbeta1 action, but not as a source of TGFbeta1. Our data indicate TGFbeta1 activity in rat pancreas extends beyond regulation of matrix production, but appears to be important in growth control of pancreatic fibroblastoid cells.
