- The Effects of High Glucose, Insulin and TGF-beta 1 on Proliferation and Differentiation of the Pancreatic Stellate Cells.
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Oak Kee Hong, Hyuk Sang Kwon, Kyu Hyun Yeom, Marie Lee, Ji Hun Yang, Seung Hyeon Ko, Soon Jib Yoo, Hyun Sik Son, Kun Ho Yoon, Bong Yeon Cha, Kwang Woo Lee, Ho Yong Son, Sung Koo Kang
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Korean Diabetes J. 2003;27(3):228-240. Published online June 1, 2003
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 Abstract
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- BACKGROUND
Although chronic pancreatitis gives rise to fibrosis of pancreatic exocrine tissue, and type 2 diabetes is accompanied by pancreatic fibrosis, the mechanisms of fibrogenesis in the pancreas have been insufficiently studied. The activated Pancreatic stellate cells (PSC) have recently been identified in human and experimental fibrotic areas from chronic panceatitis tissues. As PSC are similar in their morphology and biochemistry to hepatic stellate cells, they are suspected to play the same role in pancreatic fibrogenesis as the hepatic stellate cells in liver fibrosis. The PSC were isolated from the rat pancreata, and mediators stimulating the proliferation and differentiation identified. METHODS: The pancreatic stellate shaped cells were isolated by a minor modification to the method described by Apte et al (ref), using a Nycodenz gradient. The isolated PSCs were confirmed by phase-contrast and by the immunofluorescence of vimentin, desmin and smooth muscle a-actin (a-SMA). The level of alpha-SMA was quantified by Western blot in the PSCs in the culture, over time, and the cell proliferation was measured by 3[H]-Thymidine incorporation. The effect of the proliferation and differentiation of the PSC were assessed in relation to D-glucose (500 mg/dL), Insulin (10 IU/mL) and TGF-beta (10 ng/mL) treatment of the culture medium. RESULTS: The stellate shaped cells from the rat pancreata grew readily in the culture. Unactivated PSCs, cultured for 3 days, had an angular appearance, contained lipid droplets, manifesting positive vitamin A autofliuorescence, and stained positively for vimentin and desmin, but negatively for alpha-SMA. Within 4~8 days of primary culturing, the PSCs were activated, the sizes and numbers of the fat droplets decreased, the cells flattened, developed long cytoplasmic extensions and expressed alpha-SMA. After 3 passages, almost 100% of the cells were positive for alpha-SMA expression, indicating a myofibroblast type of differentiation in vitro. The addition of high-glucose concentrations and insulin to the activated PSCs significantly stimulated cell proliferation (194.4+/-8.3, 175.0+/-31.0 vs. control), and when the combination of high- glucose and insulin was applied, the cell proliferation was increased to an even greater extent (247.0+/-21.8 vs. control). CONCLUSIONS: Pancreata stellate cells can be isolated, and cultured in vitro, from normal SD rats. High concentrations of glucose and insulin in culture medium activated the PSC proliferation.
- The Changes of Expression of Intermediate Flament in Pancreatic Duct Cells During Proliferation and Differentiation after 90% Pancreatectomy in Rats.
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Seung Hyeon Ko, Kun Ho Yoon, Sun Hee Seo, Jung Min Lee, Ki Won Oh, Sang Ah Chang, Hye Soo Kim, Yoo Bae Ahn, Hyun Shik Son, Moo Il Kang, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
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Korean Diabetes J. 2000;24(2):191-201. Published online January 1, 2001
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Abstract
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- BACKGROUND
Neogenesis of the beta calls from ductal cells is the main mechanism of the increased beta cell mass after partial pancreatectomy. For the transdifferentiation from the duct cells to the beta cells, de-differentiation of the duct cells is needed because duct cells are also terminally differentiated cells already. But there was no clear evidence of de-differentiation of the duct cells during duct call proliferation so far. Herein we report the changes of intermediate filament protein expression in rapidly proliferating duct cells after partial pancreatectomy for the evidence of de-differentiation of the duct cells. METHODS: 45 week-old Sprague-Dawley rats weighing 80~120 g were used. 90% partial pancreatectomy was done. Experimental animals were divided into 5 subgroups by date of killing after surgery: 1, 3, 7, 14, 30 days, Pancreas remnant was excised and immunohistochemical stain was done for pancytokeratin (Pan-CK) as a epithelial cell marker and vimentin (VT) as a mesenchymal cell marker. We observed the double stained slide with pan-CK and VT antibody using confocal microscope for costaining analysis over time. The sections were also immunostained with anti-insulin antibody for the quantification of the beta cell mass by point-counting methods. RESULTS: We observed impaired glucose tolerance and diabetes were developed affer 90% pancreatectomy. Significant increase of the weight of pancreatic remnant, beta cell and duct cell mass were observed about 14 days after pancreatectomy. We observed the co-expression of VT and pan-CK intermediate filament protein in rapidly proliferating duct cells in the area of common pancreatic duct and main duct at one day after partial pancreatectomy. 3 days affer partial pancreatectomy, VT and pan-CK costained duct cells were mainly observed in the rageneration focus of the duct cell proliferation. 30 days after partial pancreatectomy, we could not find any costaining duct calls in the remnant pancreas. CONCLUSION: The vimentin intermediate filament, a marker of mesenchymal cell was expressed in proliferating ductal cells after pancreatectomy. We could suspect that pancytokeratin and vimentin co-expression is a good marker for de-differentiation of proliferating duct cells.
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KDA
