Isolation, Cultivation Of Adult Rats Pancreatic Duct Stem Cells And Experimental Study Of Autotransplantation

ObjectiveTo investigat the experiment condition of isolation,cultivation and comprehend the direction differentiate potency of adult rats pancreatic duct stem cells in vitro. Use the cell of differentiation end transplant in abdominal of rat diabetes model, observation the change with insulin secretion and blood glucose, By it estimate the therapeutic efficacy about diabetes.MethodsThe pancreas of adult wistar rats were digested by pancreatic duct in situ perfusion collagenase V. use the mothed of ficoll400 concentration gradient centrifugation to separate pancreatic duct stem cells and cultivation in vitro. according to the feature that adherence growth about pancreatic duct stem cells broth out suspension floating islet and other confounding cell. Select the cells from the serial-subcultivation to detect the expression of CK19, Pdx-1, Nestin, insulin and glucagon gene with methods of immunofluorescence staining. Then, in surrounding of the high glucose and hepatocyte growth factor(HGF) induce location differentiation, DTZ dyeing and use ELISA staining method detection insulin secretion function.. Use the method of Streptozotocin (STZ) peritoneal injection inducing diabetic rat mode. caudal vein random blood sugar exceed 16.7mmol/L indicate modeling succeed. blest diabetic rat mode separated to two group (A and B) random. Each group 20.A group is accept islet cell transplantation(experimental group);B group is placebo inject group (control group). To choose pretransplant 1 day,post transplant 7 day,14 day,21 day,28 day measure caudal vein random blood sugar and insulin.obtained The date by statistics analysis to compared the difference with two group, and then yield conclusion.ResultsUse the method of in situ perfusion collagenaseⅤcan well-distributed digest the rat pancreatic gland like fine sand. Ficoll400 concentration gradient centrifugation to make the cell that after collagenaseⅤdigest delamination clearly. To get stem cell (middle and upper stratum) can serial subcultivation 8 generation stably in vitro circumstance. The results of immunofluorescence staining revealed that the expression of CK19, Pdx-1 and Nestin of serial-subcultivation cells were positive, and the rate of positive cells were(88.6±6.2)%,(84.6±8.6)%和(81.3±7.5)%, Respectively, while insulin and glucagon staining were negative. Induce differentiate can express insulin secretion character in vitro, Diphenylthiocarbazone(DTZ) dyeing to show brownish red color. Abdominal inject 65mg/kg weight Streptozotocin (STZ) inducing diabetic rat mode were effective.The condition of hyperglycaemia can persistence not less than one month. Blood serum insulin:A group average is 9.30±1.56 MU/L B group average is 11.41±1.52 MU/L;Blood glucose:A group average is 8.22±2.7mmol/L B group average is 12.23±3.8mmol/L SPSS13.0 statistics nalysis F血糖=5.232, P血糖<0.05; F胰岛素=3.274,P胰岛素<0.05 Have statistics significant.ConclusionThis empirical method can Harvesting high purity,multi-quantitative pancreatic duct stem cell. Artificial induction may direction differentiate for islet-like clusters and have insulin secrete function. Adequacy numbers islet-like clusters transplant to alike strain rat abdominal may to add insulin secretion and degrade blood sugar. This study can be offer theory date reference to stem cell translation for treat diabetes.