| Background and aimsDiabetes mellitus (DM) defines as a metabolic disorder of multiple aetiology characterised by chronic hyperglycemia with disturbances of carbohydrate, fat, and protein metabolism resulting from defects in insulin secretion, insulin action, or both. Because type 1 diabetes is generally the result of beta-cell destruction, transplantation of Langerhans islet is a promising strategy for the treatment of it. Recently, the one year validity rate of islet cell transplantation is near to 80%, however, lack of sufficient donor organs and the rejection limit its potential. The recent success of the Edmonton Protocol for pancreatic islet transplantation has sparked new interest in transplantation of insulin-producing cells. Edmonton Protocol included, trans -plantation of large amounts of purified islet cells, combined with a glucocorticoid-free immunosuppressive regimen. However, the amount of donor islet is severely limited.Studies of experimental animal models have revealed that pancreatic islet contain a kind of stem/progenitor cells capable of islet neogenesis in the adult. These cells have been induced to produce functioning islets of Langerhans in vitro which containing A, B and D cells. Recently Zulewski reported that, the nestin-positive islet-derived stem/progenitor cells can differentiate into pancreatic exocrine/endocrine cells.Glucagon-like peptide-l(GLP-l), a hormone secreted from L cells of small intestine, can stimulate insulin synthesis and secretion. Further studies imply apotential role for GLP-1 in the modulation of islets development and differentiation, GLP-1 could promote islet cells growth, influence topography of islet, and convert pancreatic exocrine tumor cell lines into glucagons- and insulin- producing cells.The aim of our study is to isolate, culture pancreatic progenitor cells derived from newborn rats. The progenitor cells were proliferated to islet-like cell clusters (ICCs) by agar suspension culture, and then to observe the effect of GLP-1 -(7-36)amide on islet progenitor differentiation. The character and the function of the mature ICCs were observed. The ICCs were transplanted into the livers of diabetic model rats by infusion via portal vein, the therapeutic effects were observed. Materials and methods1. Islets were isolated from the pancreas of neonatal rats, the pancreatic stem/progenitor cells were purified and were proliferated by cell anchorage culture, then the progenitor cells were proliferated to ICCs by agar suspension culture.2. GLP-1-(7-36)amide were added in order to induce ICCs differentiation. Identification of progenitor cells and the cells after induction, by RT-PCR, in situ hybridization, immunocytochemistry. Determinated of C-peptide secretion in vitro (The glucose tolerance test).3. Intrahepatic ICCs transplantation by infusion via portal vein. About 4000 ICCs (=20 000IEQ/Kg)were infused into the livers of the ICCs transplantation group ratsvia the portal vein. Two groups were selected as control, one group was given sham transplant of saline solution, the other was injected of saline solution intraperitoneal. The blood sugar level and the survival time were monitored on subsequent days. 15 days after surgery, the livers were all collected for immunohistochemical analysis. Results1. The nestin-positive pancreatic stem/progenitor cells could be isolated from the pancreas of neonatal rats, which could be passaged 12 passages in vitro, and could be proliferated to islet-like cell clusters (ICCs).2. The islet progenitor cells did not express insulin mltf"^ CK—19 ^ PDX-1 ^ insulin and somatostatin, but nestin, before differentiation by GLP-l-(7-36)amide.After differentiation, the percent of positive cell expressed insulin mRNA, CK—19, PDX-1, insulin, somatostatin and nestin was62.0%, 0%, 72.0%, 62.5%, 6.5% and 5%.RT—PCR results. The expressions of pdx-1 mRNA were significant increased after differentiation.Radioimmunoassay results. C—peptide concentrations in normal media was 0.076±0.007 pmol/ml. C—peptide concentrations in media after 2 weeks, 3 weeks of differentiation, were 1.225±0.135 pmol/ml, 5.386±0.628 pmol/ml.The glucose tolerance test of ICCS in vitro results. C—peptide concentrations of Hyperglucose+Aminophylline group, Hyperglucose group, Hypoglucose group, were as follows: 2.616 ±0.140 pmol/ml, 1.606 ±0.085pmol/ml, 0.776 ±0.051 pmol/ml. The difference was statistic significant among three groups (iM).O5) .3. Before transplantation, the blood sugar in the ICCs transplantation group rats was all higher than 16.7 mmol/L. However, it was declined 3 days after transplantation. Before and after transplantation, the difference was statistic significant (P<0.05) . The insulin positive cells could be found in livers of the ICCs transplantation group rats. Conclusions1. A kind of islet progenitor cell exists in pancreatic islet of neonatal SD rats, which could be expanded continuously in vitro by agar suspension culture.2. GLP-1 can differentiate the progenitor cells into ICCs capable of secretion.3. The ICCs can reverse hyperglycemia of diabetic model rats, after they were transplanted into the livers by infusion via portal vein. |
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