| Diabetes mellitus is a devastating disease all over the world. With the increasing morbidity of diabetes mellitus,the diabetes mellitus has now become the third highest incidence following cardiovascular disease and tumour which are chronic diseases heavily threatening people's common health.Its syndrome caused by hyperglycosemia put heavy burden on patient not only in economic but also in spirit.Diabetes mellitus has a increase trend.In our country,its incidnce in 1994 asend to 3 times to 1980.and it was estimated that diabetes will increse to 1 billion in following 30 years and its incidence will be 5% to 10% of the total number of people.Thus, it is urgent studing on prevention and treatment methods for Diabetes mellitus.The general principle of diabetes therapy is to supplied with insulin rapidly, availably and chronically to replenish the self-secreting lack of insulin.Restablishing a endogenic insulin secret system is the best way to fufil these demand.The pancreatic stem cells can be unlimitly proliferated and differantiated into pancreatic endocrine cells.if we get this multipotential cells easily and directed its differentiation according to patient's need.that should give hope to diabetes therapy.Following former researcher methods,pancreas collected from 90-day old porcine fetus was dissected and then digested with 0.25% trypsin for 10 min. The primary cells were cultured in medium of RPMI 1640 with 10%FBS (added 1mmol/L pyravate, 71.5μmol/Lβ-Me, 20ng/ml EGF,20ng/ml bFGF).the cell derived from porcine fetus were identified as pancreatic stem cell by immunochemical method.During cells cultivation and passage,we have found that the pancreatic stem cell couldn't be passed over 8 time only in 10% serum concentration. Low concertration of serum made most of the cells died, only a few cells survived that have the small and round morphology attached to the surface of culture plate.But we found if we changed medium to RPMI 1640 with 20% FBS (supplement with 1 mM pyravate, 71.5μMβ-Me, 20 ng/ml EGF, 20 ng/ml bFGF) at this time ,these little cells started to become bigger and proliferate to form clusters. It grew to confluence in another 6 days. After the first few passages, cell morphology was diversified into mesenchymal-like and fibroblast-like cells. Four cell strains were finally purified by this method. One of the strains was continually grown for 64 passages in 14-month culture period and presented the immortalized feature. The results of immunochemistry study showed that the isolated cell expressed pancreatic duodenal homeobox-1 (PDX-1), glucose transporter-2 (GLUT-2) , Proliferating cell nuclear antigen (PCNA),nestin and vimentin biomarkers that were thought as markers of the pancreatic stem cell.To induce cell differentiation,cells grown in medium of DMEM/F12 with 2% B27,0.5%BSA,10mM nicotinamide,5ng/ml HGF,10ng/ml EGF,20nM GLP-1,100U/ml Penicillin/Streptomycin Solution for 6-8 days were formed the islet-like cell clusters) The results of immunochemistry study showed that the isolated cell retained PDX-1,GLUT-2 and vimentin biomarkers when cells forming the cluster. After differentiation, several endocrine hormones that were specially produced from pancreatic islet cells were also detected in the cell that included insulin, glucagon, somatostatin and polypeptide. This investigation revealed that the cell was the pancreatic stem cell and was able to differentiate to all four kinds of pancreatic islet like cells in vitro.we also found the viability of formed ICCs were not good in induction medium without serum,so we added serum in induction medium,the results revealed that the pancreatic stem cells can also differentiated into endocrine cells in induction medium with serum and retain good viability. By means of radioimmunoassay(RIA),βcells induced from the pancretic stem cell had an increase in secretion of insulin and c-peptide. The singleβcelI was also brightred for dithizone(DTZ) staining.These results indicated that there are multipotent stem cells in pancreas. The present study offers an approach to isolate and expand pancreatic stem cells from porcine fetus, and differentiate them into functionalβcells in vitro. It likely become an new source for diabetes therapy through xenotransplantation of pancreatic stem cells.The immunohistochemical staining also showed that the isolated cells express the markers of the embryonic stem cell including SSEA-1 and Oct4.at the same time,cells were positive for AKP, which indicated that the cells isolated possess the multipotential properties. Prior to treating with medium supplemented 5% NBS and 1 mmol/Lβ-mercaptoethanol(β-Me) for 24h, the isolated cells were treated with 5 mmol/Lβ-Me, no serum for 4h sequentia to differentiate toward neural cells. The induced cells expressed NF,the marker of neural cells.Differentiation of isolated cells toward the osteoblast lineage can be induced by supplementing 0.1μmol/L dexamethasone, 10 mmol/Lβ-glycerophosphate and 50μg/mL ascorbic acid. The cells were induced forming cluster after 7 day's culture.The cells were positive stained with alkaline phosphatase (AKP).Cells were injected into groin of nude mouse at 106,tumour were not found that imply the pancreatic stem cell has no oncogenesis。We injectedβcells induced from the pancretic stem cell into cavum abdominis of rat which were diabets modle treated by STZ(streptocizone),hyperglycemia of rat were not be remited during the period of 60 days treatment.that imply rejection reaction was the obstacle for apply the pancretic stem cell to therapy,its effect should be satisfied if its rejection reaction were decresed.In conclusion, four individual pancreatic stem cells were isolated from porcine pancreas tissue in which one cell strain was continually cultured for 14 months (to 64 passages). This study provided a simplified procedure to isolate and culture pancreatic stem cells from animal pancreas, and established an animal cell strain for cell therapy in human diabetic mellitus.
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