| Diabetes mellitus is a devastating diseases with severe secondary complications driven by poor glycaemic control. Patients with diabetes depend on daily injections of exogenous insulin for regulation of glucose homeostasis. Insulin, however, is not a cure and does not perfectly simulate the strict control of blood glucose that is provided by theβcells and therefore may cause hypoglycemia. Islet transplantation has been impressive in rendering some patients insulin-independent for a number of years, but the promise of allogeneic islet transplantation has been hindered by the limited supply of islets. Researchers now search for the replaceable resources, such as stem cells or porcine islets. Pancreatic stem cells are adult stem cells which harbors in the pancreas and retain the ability of self-renewal and multiple differentiation. Pancreatic stem cells have a number of advantages over other stem cells when induced to differentiate into islet cells, such as the easier method, the higher successful rate, the lower risk of oncogenesis, without the ethical controversies and so on. They hold promise in the treatment of diabetes.A cell line of human fetal pancreatic stem cells was established in our lab in 2004. But the characteristics of the pancreatic stem cells were not identified clearly. The aim of this research is to study the features of the pancreatic stem cells systematically through their markers at both protein and mRNA levels, growth curve, and differentiation ability both in vivo and in vitro; we also induce pancreatic stem cells to differentiate into islet-like cell clusters and discuss their effects on reversing diabetes in diabetic rats by transplantation. The main contents are as follows:1. Characteristics of human pancreatic stem cellsThe results of immunohistochemical staining showed that the pancreatic stem cells expressed PDX-1, Nestin, CK7, CK19, glucagon, somatostatin, pancreatic polypeptide, GLUT2, E-Cadherin, vimentin, AFP and PCNA, and did not express insulin orβ-Amyloid; the results of RT-PCR further confirmed that the cells transcripted PDX-1, Nestin, CK7, CK19, GLUT2 andβ-Gal, while not the mRNA of insulin; the results of FACS analysis showed that the cells expressed CD29, CD44, CD166, while not expressed CD14, CD34, CD45, CD11a, CD90, CD105, CD117. Taken together, all these results suggested that the expression features of the pancreatic stem cells were similar with those reported by other researchers.The pancreatic stem cells grew clonally. They are epithelioid when plated at a low density; they formed a slabstone-like monolayer when they reached 70% confluence. The growth curve showed that the pancreatic stem cells grew slowly in cultured 1-4 days and stayed in latent period, and entered the logarithmic growth period when cultured 5-7 days, then they grew slowly. Compared with the 33rd and 44th cells, the 20th cells grow more quickly from the 6th day. The pancreatic stem cells have been maintained for 3 years.Grafts formed at groin after the pancreatic stem cells had been transplanted into the nude mice for 6 weeks. The results of H.E staining and immunohistochemical staining suggested that the cells could differentiate into pancreatic cells spontaneously in vivo. The pancreatic stem cells could be induced in vitro into DTZ-positive pancreaticβcells by serum-free medium contained bFGF and HGF ,β-tublin-positive neural cells byβ-Me, Toluidine Blue-positive chondrocytes by dexamethasone. But the cells could not be induced to differentiate into myocardial cells by 5-Aza, which suggested that the condition may not be suitable for their differentiation into myocardial cells or they may have a limited differentiation potential. Taken together, all these results suggested that the pancreatic stem cells have the potential to differentiate into cells from the three different layers.2.Differentiation of human pancreatic stem cells into islet-like cell clusters in the treatment of diabetic ratsHuman pancreatic stem cells differentiated into islet-like cell clusters when cultured in serum-free differentiation medium. The islet-like cell clusters were stained red by dithizone (DTZ), expressed insulin mRNA by RT-PCR and secreted insulin with high glucose stimuli. They were transplanted into the left subrenal capsule of the diabetic rats which experimental diabetes was induced by a single intraperitoneal injection (70mg/kg of body weight) of streptozotocin (STZ). After 48h of transplantation a decrease in blood glucose was observed and blood glucose levels remained lower in comparison with their diabetic counterparts for 2 weeks and then rose to higher levels. The result suggested that islet-like cell clusters generated in vitro from human pancreatic stem cells had some effects on reversing the diabetes in diabetic rats.
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