Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization.
OBJECTIVE
Activated pancreatic stellate cells (PSCs) have been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Primary PSCs can be subcultured only several times because of their limited growth potential. A continuous cell line may therefore be valuable in studying molecular mechanisms of these pancreatic disorders. The aim of this study was to establish a cell line of rat PSCs by spontaneous immortalization.


METHODS
PSCs were isolated from the pancreas of male Wistar rats, and conventional subcultivation was performed repeatedly. Telomerase activity was measured using the telomere repeat amplification protocol. Activation of transcription factors was assessed by electrophoretic mobility shift assay. Activation of mitogen-activated protein (MAP) kinases was examined by Western blotting using anti-phosphospecific antibodies. Expression of cytokine-induced neutrophil chemoattractant-1 was determined by enzyme immunoassay.


RESULTS
Conventional subcultivation yielded actively growing cells. One clone was obtained after limiting dilution, and designated as SIPS. This cell line has been passaged repeatedly more than 2 years, and is thus likely immortalized. SIPS cells retained morphological characteristics of primary, culture-activated PSCs. SIPS expressed alpha-smooth muscle actin, glial acidic fibrillary protein, vimentin, desmin, type I collagen, fibronectin, and prolyl hydroxylases. Telomerase activity and p53 expression were negative. Proliferation of SIPS cells was serum-dependent, and stimulated with platelet-derived growth factor-BB through the activation of extracellular signal-regulated kinase. Interleukin-1beta activated nuclear factor-kappaB, activator protein-1, and MAP kinases. Interleukin-1beta induced cytokine-induced neutrophil chemoattractant-1 expression through the activation of nuclear factor-kappaB and MAP kinases.


CONCLUSIONS
SIPS cells can be useful for in vitro studies of cell biology and signal transduction of PSCs.
Masamune
Atsushi
A
Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan. amasamune@int3.med.tohoku.ac.jp
Satoh
Masahiro
M
Kikuta
Kazuhiro
K
Suzuki
Noriaki
N
Shimosegawa
Tooru
T
D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000473:pathology; D003598:Cytoskeletal Proteins / Q000032:analysis; D016326:Extracellular Matrix Proteins / Q000032:analysis; D007150:Immunohistochemistry; D016328:NF-kappa B / Q000235:genetics; D015345:Oligonucleotide Probes; D010179:Pancreas / Q000166:cytology* / Q000473:pathology; D051381:Rats; D018808:Transcription Factor AP-1 / Q000235:genetics
D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D018808:Transcription Factor AP-1