| Part I Isolation, cultivation and identification of adult rats pancreatic duct stem cells in vitroObjective To explore the method of isolation, cultivation and identification of adult rats pancreatic duct stem cells in vitro. Methods The pancreas of adult rats were digested by injecting collagenase V solution and purified by removing the islets with Ficoll density gradient centrifugation .The islet-depleted tissue was cultured in RPMI1640 with 10% Fetal Calf Serum, then the medium was supplemented with EGF(epidermal growth factor) and bFGF(basic fibroblast growth factor) for 7 days. The cells could form a monolayer, 0.25% Trypsin-EDTA were used to detach the cells and passaged. Cells from the second passage (P2) were used to detect the expression of CK19, Pdx-1, Nestin, insulin and glucagon with methods of immunofluorescence stain and RT-PCR.Results Through collagenase solution injecting and digesting the pancreatic duct, and depleting the islet tissue after Ficoll density gradient centrifugation, the pancreatic duct cells could get a better purification. The results of Immunofluorescence stain revealed that the expression of CK19, Pdx-1 and Nestin of P2 cells were positive and the rate of positive cells were( 88.6±6.2 )%, (84.6±8.6), (79.3±10.5)%, respectively, while insulin and glucagon stain were negative. The results of RT-PCR demomstrated that the cells also had the expression of CK19, Pdx-1, Nestin.Conclusions This method can isolate the pancreatic duct cells well, and through identification, the cells obtained have the character of stem cells.Part II Isolation, cultivation and identification of adult rats pancreatic duct stem cells in vitroObjective To establish a new protocol for differentiation pancreatic stem cells into insulin-producing cells with islet neogenesis-associated protein (INGAP ) in vitro.Methods The pancreatic duct stem cells were isolated and cultured in vitro. Cells from the second to six passages were used to differentiation. Then the cells were divided into two groups in the anaphase of cell division, one cultured in the medium supplemented with INGAP-related pentadecapeptide and the other with control peptide. The new produced islet-like clusters (ILCs) were harvasted when the end of the differentiation and detected by immunofluorescence stain and RT-PCR. Meanwhile, the ability of insulin secretion was evaluated throgh glucose stimulated insulin secretion (GSIS).Results The pancreatic duct stem cells from the second to six passages could thansform to ILCs which had spherical structure after differentiated with INGAP-related pentadecapeptide by a four-step method. The results of immunofluorescence stain revealed that insulin and glucagon stain of the ILCs were positive. The results of RT-PCR revealed that the ILCs also had the expression of insulin and glucagon. The correspondence expression amout of insulin: INGAP group was 0.324±0.09, control group was 0.102±0.04, there was significant difference between the two groups (P <0.01 ) ; The correspondence expression amout of glucagon: INGAP group wasO.260±0.05, control group was 0.085±0.04, there was also significant difference between the two groups (P<0.01) . When compared to normal islets, the correspondence expression amout of insulin and glucagon of INGAP goups was not significant difference. The stimulated index in low glucose and high glucose: INGAP group was(3.3±0.1), control group was(2.6±0.4), nomal islets was(3.1±0.4); Compared to control group, The secretion amount of insulin at low and high concentration of glucose was significantly increased in INGAP group (P<0.01) .Conclusions Through the new protocol with INGAP-related pentadecapeptide we can obtain the new produced islet-like clusters that possess spherical structure and insulin secretion ability, this studyprovides a more effective method to obtain new sources ofβcells forislet thansplantation.
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