The Research Of Immunological Characteristics Of Pancreatic Stem Cells And Transdifferentiated Process

Objective: To isolate, purify and expand the rat pancreatic ductal epithelial cells, then,induce them into pancreatic islet cells and to isolate, purify normal islet cells.Compare pancreatic stem cell and the induced islet cells with the normal islet cells inimmunology characteristic, to investigate immunological problem of pancreatic stemcell in the clinical application, to provide basic for pancreatic stem cell in the clinicalapplication.Methods: (1) Pancreatic ductal epithelial cells were isolated and purified, then wereidentified with CK-19 by immunocytochemical stain. At the stage of 80％-90％confluency, pancreatic ductal epithelial cell cultures were set in serum-free mediumwhich included exendin-4,KGF,NIC.(2)To observe the change of cell, inducedislet like cell clusters within four weeks were confirmed by dithizone staining.(3)Toisolate and purify normal islet cells were confirmed by dithizone staining.(4)Separatesrat lymphoid blood to culture with pancreas stem cell and induced islet like cell andnatural islet respectively, examine IL-2 secretion with the ELISA, IFN-γ, secretionwith the Elispot examination, the expression of MHC-Ⅰand MHC-Ⅱof lymphoidcell after co-culture by the flow cytometry.(5)Infuse pancreas stem cell, induced isletlike cell and natural islet into the rat thigh seamy side muscle respectively, sentencesthe rats after 5 days, doing the pathologic dyeing of HE to view its Inflammatoryreaction.(6)Build 1 diabetes model of rat, infuse pancreas stem cell, induced islet likecell and natural islet under the rat skin respectively, in 1d, 3ds, 5ds, 7ds sampling therat peripheral blood flow examining the MHC-Ⅱof expression and the IFN-γsecretion in serum with ELISA.Result: (1) After isolation and purification, the pancreatic ductal epithelial cellscould be proliferated efficiently; most cells expressed CK-19. (2)After four weeks,there was movement of the epithelial cells into islet like cell structures that DTZstaining was positive. (3) To isolate and purify normal islet cells were confirmed thatDTZ staining was positive. (4) The density of the IL-2 that the lymphoid cell secreted about 44.1ng/ml, 230.7ng/ml and 317.6ng/ml, the spot of IFN-γsecreted increasesone by one in order, the expression of the MHC-Ⅰdistinguishes to 15.0％, 16.7％,17.9％, the expression of the MHC-Ⅱabout is 0.8％, 29.5％, 32.6％respectively.(5)The pathologic HE dyes manifestated: pancreas stem cell, induced islet like cell andnatural islet, inflammatory reaction to strengthen one by one in order. (6)Build themodel success, pancreas stem the cell showed no MHC-Ⅱexpressions difference, butthe induced islet like cell and the natural islet that induce, the expression of the MHC-Ⅱat any time an extension but increase. The examination of the IFN-γto serum, theinduced islet like cell and the natural islet had obviously higher IFN-γthan thepancreatic stem cells.Conclusion: We have been able to expand rat duct epithelial cells that have the stemcell potential efficiently.The comparison of the immunology characteristic of naturalislet and pancreatic stem cells indicated that the pancreatic stem cells had obviouslylower immunogenicity. The comparison of the immunology characteristic of naturalislet and the induced islet like cell indicated that islet like cell had gradually toheighten. The result indicated that immunogenicity had gradually to heighten in theprocess of pancreatic stem cells Tran-differentiated islet cell, to provide the basis ofthe immunologic response of remedial stern cell.