AIM: 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 pote...AIM: 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 α-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-1β activated nuclear factor-κB, activator protein-1, and MAP kinases.Interleukin-1β induced cytokine-induced neutrophil chemoattractant-1 expression through the activation of nuclear factor-κB and MAP kinases.CONCLUSION: SIPS cells can be useful for in vitro studies of cell biology and signal transduction of PSCs.展开更多
OBJECTIVE: To investigate whether the decrease in expression of interleukin-2 (IL-2) after trauma is associated with changes in DNA binding activity of nuclear factor of activated T cells (NFAT) and activator protein-...OBJECTIVE: To investigate whether the decrease in expression of interleukin-2 (IL-2) after trauma is associated with changes in DNA binding activity of nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1). METHODS: Mice with closed impact injury with fracture in both hind limbs were adopted as the trauma model. Spleen lymphocytes were isolated from traumatized mice and stimulated with Con-A. Culture supernatants were assayed for IL-2 activity, and total RNA was extracted from spleen lymphocytes and assayed for IL-2 mRNA. DNA binding activity of NFAT and AP-1 were measured by electrophoretic mobility shift assay (EMSA). The expression of c-Fos, c-Jun and JunB proteins was determined by the Western blot analysis. RESULTS: DNA binding activity of NFAT and AP-1 gradually decreased to a minimum of 41% and 49%, respectively, of the control on the 4th day after injury, which was closely followed by the decline in IL-2 activity and IL-2 mRNA. A decrease in the expression of c-Fos on the 1st and 4th day after trauma had no significant effect on c-Jun expression; the increase in expression of JunB was only on the 1st day after injury. CONCLUSION: Decreased IL-2 expression is, at least in part, due to a decline in the activation of NFAT and AP-1 in traumatized mice. The decline in DNA binding activity of NFAT and AP-1 is partly due to a trauma-induced block in the expression of c-Fos.展开更多
基金Grant-in-Aid for Encouragement of Young Scientists from Japan Society for the Promotion of Science(to A.M.)Pancreas Research Foundation of Japan(to A.M.)
文摘AIM: 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 α-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-1β activated nuclear factor-κB, activator protein-1, and MAP kinases.Interleukin-1β induced cytokine-induced neutrophil chemoattractant-1 expression through the activation of nuclear factor-κB and MAP kinases.CONCLUSION: SIPS cells can be useful for in vitro studies of cell biology and signal transduction of PSCs.
文摘OBJECTIVE: To investigate whether the decrease in expression of interleukin-2 (IL-2) after trauma is associated with changes in DNA binding activity of nuclear factor of activated T cells (NFAT) and activator protein-1 (AP-1). METHODS: Mice with closed impact injury with fracture in both hind limbs were adopted as the trauma model. Spleen lymphocytes were isolated from traumatized mice and stimulated with Con-A. Culture supernatants were assayed for IL-2 activity, and total RNA was extracted from spleen lymphocytes and assayed for IL-2 mRNA. DNA binding activity of NFAT and AP-1 were measured by electrophoretic mobility shift assay (EMSA). The expression of c-Fos, c-Jun and JunB proteins was determined by the Western blot analysis. RESULTS: DNA binding activity of NFAT and AP-1 gradually decreased to a minimum of 41% and 49%, respectively, of the control on the 4th day after injury, which was closely followed by the decline in IL-2 activity and IL-2 mRNA. A decrease in the expression of c-Fos on the 1st and 4th day after trauma had no significant effect on c-Jun expression; the increase in expression of JunB was only on the 1st day after injury. CONCLUSION: Decreased IL-2 expression is, at least in part, due to a decline in the activation of NFAT and AP-1 in traumatized mice. The decline in DNA binding activity of NFAT and AP-1 is partly due to a trauma-induced block in the expression of c-Fos.
