Dissertation
Dissertation > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetes

Isolation、Incubation and Differentiation of Pancreatic Islet-Derived Progenitor Cells from Newborn SD Rats

Author WangQingYong
Tutor YaoYunWei
School Zhengzhou University
Course Physiology
Keywords pancreatic islets stem/progenitor cells nestin GLP-1(7-36)NH2 differentiation
CLC R587.1
Type Master's thesis
Year 2004
Downloads 108
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Background and aims: Diabetes mellitus(DM) defines as a metabolic disorder of multiple aetiology characterised by chronic hyperglycaemia with disturbances of carbohy- drate, fat, and protein metabolism resulting from defects in insulin secretion, insulin action, or both. Diabetes is associated with several microvascular complications, including neuropathy, nephropathy, and retinopathy, as well as macrovascular complications such as myocardial infarction, stroke, and peripheral vascular disease. Type 1 diabetes is generally the result of beta-cell destruction, transplantation of Langerhans islet is a promising strategy for the treatment of it. Only about 10% of the patients was insulin independent a year after receiving a transplant in past. The recent success of the Edmonton Protocol for pancreatic islet transplantation has sparked new interest in transplantation of insulin-producing cells. Edmonton Protocol include, transplantation of large amounts of purified islet cells, combined with a glucocorticoid- free immunosuppressive regimen. Seven patients showed normalisation of glycated haemoglobin concentration and lasting independence from insulin injections for 1 year, however, the amount of donor islet is severely limited. Stem cells, a unique cell population with the ability to undergo both self-renewal and differentiation, could provide a alternative source of islet tissue. At present, stem cells can be indentified in many adult mammalian tissue, such as bone marrow, blood vessels, skeletal muscle, cornea, retina, liver, brain, epithelia of the skin and digestive system.Studies of experimental models of pancreas injury have revealed that pancreatic islet contain a kind of stem/progenitor cells capable of islet neogenesis in the adult. Recent reports have demonstrated the capacity of nestin-positive islet- derived progenitor cells both from human fetal and rat adult pancreas to differentiate into insulin-producing cells in culture. Glucagon-like peptide-l(GLP-l), a hormone secreted from L cells of small intestine, can stimulates insulin synthesis and secretion. Recent studies imply a potential 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 aims of the present study were to isolate, culture pancreatic progenitor cells derived from islets of newborn rats, and then to observe the effect of GLP-1 (7-36) NH2 on islet progenitor differentiation.Materials and methods: Isolation and purification of islets 1-3 day-old Sprague-Dawley rats were killed by decollation, pancreases were removed and dissected into 0.5mm3 segments and digested with 10ml lg/L collagenase V for 20-30 minutes at 37C. Islets were washed three times with cold Hank’s balance salt solution. Thoroughly washed islets were suspended in modified RPMI-1640 medium (11.1mmol/L glucose) supplemented with 10% fetal bovine serum, 10 mmol/L Hepes, 1mmol/L sodium pyruvate, 100,000IU/L penicillin and 100mg/L streptomycin, and placed into 50cm2 flasks. The islets were incubated for 20 hours at 37C with 95% humidified air and 5% CO2. After 20 hours of incubation, the medium containing the suspended islets were carefully removed and washed with serum-free medium, then resuspended and incubated in RPMI-1640 medium supplemented with 2.5mg/L acetic iodine for 5 hours. The purified islets were washed three times with serum-free medium.Proliferation and differentiation of islet progenitor cells The purified islets were resuspended in the modified RPMI-1640 medium supplemented with 20 ug/L bFGF and 20ug/L EGF, added into 25cm2 cell culture flasks, every flask contain 20-30 islets.. The islet adhered to substrate immediately, a monolayer of shuttle cells grewout and away from the islets. Cells in suspension were rinsed and removed with sucker. Shuttle cells, namely, islet progenitor cells, were expanded repeatly in growth factor-supplement and passaged

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