Studies On Isolation, Culture And Identification Of Islet Stem Cells And Its Biological Functions In Mice

Diabetes is prevalent all over the world. There are three key therapeutics fordiabetes, the first way is insulin injection in vitro, second is pancreas transplantation,third is islets transplantation. Nowadays, insulin injection is the most common wayto cure diabetes, but long time insulin injection make patient daily lifeuncomfortable and cause other neopathy, such as nephropathy and angiocardiopathy.Although pancreas transplantation and islets transplantation can also cure diabetesand the patient no need to inject insulin every day, these ways have some flaw thatcause them can not be used like insulin injection, like as low success rate, expensive,high risk and so on. The most factor restrict these two therapeutics cure diabetes isthe shortage of donor. In general condition, one diabetes patient needs at least twohuman cadaveric pancreases. In order to make such a therapy available to more thana few of the thousands of patients with diabetes, new sources of insulin-producingcells must be identified. The recent conceptual stem cells in bone marrow and inmost, if not all, organs suggests that adult stem cells may be potential sources of newislet cells. But no clearly identifiable adult pancreatic stem cell has been found as yet,but there is considerable evidence that such cells exist and several candidate cellshave been reported. Such as nestin positive cells in islets, pancreatic ductal cells,c-Met positive cells, carbonic anhydrase Ⅱ positive pancreatic cellshave been showto have the identity of βcells progenitor. In2003, Humphrey proved that nestin is nota specific marker of β-cell precursors in the developing human pancreas, this resultis different with the found of Zulewski, in2001.We used two ways to isolate islets from ICR mouse, dissected collagenasetechnique and intraductal collagenase technique. The result show that, at the same digestion condition (collagenase concentration0.5mg/ml,37℃,20min), theintraductal collagenase technique（105±8） is more effective than the dissectedcollagenase technique（47±8）. Then we improved the digestion condition for theintraductal collagenase technique, found that the best digestion condition iscollagenase concentration0.8mg/ml,37℃,20min(138±11). Using DTZ stain andinsulin immunofluorescence, results show that the isolated islets quality is fine. Nextstep, we found exist of nestin positive cells in islet of ICR mouse through RT-PCRand nestin immunofluorescence technique. Then we isolation, culture andidentification of those nestin positive cell in vitro. Insulin immunofluorescenceresults show that those primitive nestin positive cell do not express Insulin gene. Aslong as the isolated nestin-positive islet-derived progenitor cells (NIPs) areexpanded in the presence of basic fibroblast growth factor (bFGF), epidermal growthfactor(EGF),11.1mmol/L glucose,10%serum and are not allowed to becomeconfluent, they proliferate and express nestin. But when expanded in2.5mmol/Lglucose,2%serum, without bFGF and EGF and are allowed to become confluent,those nestin positive cells can form islet-like structure and express Insulin gene. Theislet-like structure show mahogany after DTZ staining, the similar with islets afterDTZ staining.Those results indicated that the exist of nestin positive cells in ICR mouse islet,and those nestin positive cells have the biological properties of pancreas precursorcells.