Separate purification and directed differentiation potency of pancreatic stem cells of adult rats in vitro / 中华胰腺病杂志

ABSTRACT

Objective To investigate the experimental condition of separate purification and directed differentiation potency of pancreatic stem cells of adult rats in vitro. Methods The pancreas of adult rats were digested by collagenase V in situ perfusion. Filtration was performed by 100 mesh filter. Ficoll 400 density gradient centrifugation was used to separate pancreatic stem cells and stem cells were for cultivation. Epidermal growth factor (EGF), fibroblast growth factor (bFGF) was added to medium for in vitro cultivation.Immunofluorescence staining method was used to detect the expression of CK19, Pdx-1, nestin, insulin and glucagon, and the positive cell rate was calculated. The differentiated cell was evaluated by dithizone (DTZ)staining; insulin excretion function was determined by optical enzyme labeled ELISA staining. Results The pancreatic stem cell obtained from the study could be cultivated in vitro for 8 generations. The expression of CK19, Pdx-1 and nestin of the tem cells were all positive, and the rate of positive cells were (88.6 ± 6.2)% ,(84.6 ±8.6)% and (81.3 ±7.5)%. The differentiated cell was in brownish red color by DTZ dye. After high concentration sugar stimulation, the expression of insulin secretion was increased in the supernatant.Conclusions This method can harvest highly purified, and large amount of pancreatic duct stem cell.Artificial induction may result directed differentiate for islet-like clusters and have insulin secrete function.