The Isolation And Culture Of Islets Langerhans And Islet Stem Cells In Rat

Stem cells retaining the ability of proliferation and differentiation have been identified in adult tissue. Because β-cells in pancreas belong to the end-stage differentiation cells, and have low ability of proliferation, most investigators believe that neogenesis of β-cellls rely on the proliferation and differentiation of stem cells in adult. This neogenesis can be found in the animal-model of partial wrapping pancreatic duct. Pancreatic stem cells have been isolated and cultured in vitro duo to the development of the technique of cell culture, but the characteristic of stem cell has not been well clarified, as a result , the insulin release level from the β-cells deriving from pancreatic stem cells is very low, and far away from clinic use. In this study nestin positive-cells were isolated and cultured from rat non-islet cells in vitro, and could form islet –like cell mass that had the function expressing insulin. From this study it can be concluded that pancreatic nestin positive cells possess the characteristic of stem cells, and deserved to be studied further.Part Ⅰ:The isolation of islet from neonatal rats and adult rats. The isolation of islet of neonatal rats was completed by the method we modified. The original method of isolating neonatal rat islet was to remove and dissect pancreas into small segments, then to be digested by collagenase. The islet obtained by the original method showed , most of the cases, damage both in morphology and function. Because there are little fibrous tissue in neonatal rat pancreas, we put the whole pancreas into collagenase at a concentration of 1mg/ml to digest under 37℃. After 14min, the pancreas turned into small particle, containing islets obtained with better morphology and function. The opportunity of contamination was greatly reduced. The isolation of adult rat islet was accomplished with the method used by Ricordi and his coworkers.PartⅡ:The culture of rat pancreas stem cells in vitroThe islet obtained from rat pancreas was suspended in modified RPMI1640 media(10% fetal bovine serum,PH7.6) and incubated under 37℃ with 5%CO2. After 36h,some cells adhered to the surface of culture plates, but islets were not adhered ,then removed the liquid, and added RPMI1640(free serum, pH7.4, add bFGF, EGF each 20ng/ml,1%N2). After 4-7 days, a new formed monolayer of cells could be observed, which continued to form Islet-Like Cells Mass(ILCM) 18-22 days later. After adding nicotinamide, the number of Islet-Like Cells Mass increased obviously, and expressed insulin that could be detected by 131I immunoradiometric assay. The islet-like cells mass could be passaged several times. But the number of Islet-Like Cells Mass was fewer in adult rats compared with neonatal rats.The Islet-Like Cells Mass possess the ability of proliferation and differentiation that is similar with IPSCs and CHIBs reported by Ramiya and Bonner-Weir. But Zulewski reported that nestin positive cells existed in islet and possessed the similar quality. In order to find the origination of Islet-Like Cells Mass, further study is necessary.Part Ⅲ: The distinction of ILCMImmunofluorescytochemistry method was used to detect CK19, insulin, glucagon and nestin in monolayer of cells, it was founded that most monolayer of cells could express nestin, but could not express CK19, insulin, and glucagon e. After passage, the Islet-Like Cells Mass could express nestin,insulin, and glucagon. We concluded that the adhered cells might be nestin positive cells , which possess the characteristic of stem cells. To detect CK19 and nestin, the total cellular RNA abstracted from monolayer cells was transcripted reversely and amplified by polymerase chain reaction. The primers of CK19 and nestin were designed according to that reported by Zulewski. After 35 cycles amplify, we obtained a DNA product (523bp) which was confirmed by genebank as nestin, but not CK19 products, suggesting that the adhered cell was nestin positive cells, other than CK19 positive cells. CK19 and nestin were th...