| BackgroundDiabetes mellitus is one of the principal diseases affecting mankind.The therapies include the traditional drugs,injection of insulin,pancreatic organ transplantation and islets transplantation.The pancreas transplantation is a complicated technique, perioperative complications incidence is high,and the operation indication is still limited to the patients suffered from diabetes in combination with renal failure who need to receive combined pancreas and kidney transplantation,which is currently performed in the large center of transplantion across the world.The cells substitution therapy represented by islets cells transplantation has the advantages of simple operation and minimal invasion.Compared to the injection of insulin,islets grafts can mornitor the blood glucose timely,and release the insulin in time,which keep the body glucostasis and prevent diabetic nephropathy and retinopathy from happening. In 1999,the applications of "Enmonton Protocol" significantly increase the therapeutic effect of islets tranplantaion,which is the milestone of modern islets tansplantion.However islets transplantation confronts the problem of limited donors, plus the mass mortality and functional lesion of islets damaged by perioperative technique and change of circumstance,which finally limited the wider application of islets transplantation.So it becomes the hot topic recently in the field of islets transplation to enlarge the sources of islets cells.Some authors tried to adopt heterogeneity islets transplantation(like pigs)to resolve the insufficiency of islets donors,and they also believed this strategy may avoid the autoimmune attack of type 1 diabetes mellitus.However most scientistes consider this technique can be developed only human understand the mechanism of amphixenosis because it has the potential dangers of retro virus infection.To resolve the tough problem of islets donors' shortage,the research of stem cells are becoming the hot topic recently.The most obvious biological characteristics of stem cells are to possess the self-renewal and proliferative ability,and also the potency of multi-directinal differentiation.Stem cells can be divided into embryonic stem cells and adult somatic stem cells according to the source.Pancreatic stem cells belong to adult somatic stem cells and don't have the exact definition yet.It generally refers to the indeterminate cells before terminal differentiation that can generate islets tissue or origin from islets and possess self-renewal.Pancreatic stem cells belong to the same cell lineage with pancreatic endocrine cells,so the process of differentiating into target cells is the shortest,which get pancreatic stem cells the promising source of islets transplantion.Other advantages include(1)high proliferation and potency of multi-directional differentiation,which can completely resolve the problem of islets shortage,(2)avoidance of immunological rejection via pancreatic biopsy to gain autologous pancreatic stem cells,establishment of stem cells treasury,and genetic engineering to reform or modify homologous pancreatic stem cells,(3)avoidance of tumor resulted from embryonic stem cells proliferation,and(4)avoidance of social ethical issues brought by using embryonic stem cells.At present many authors have explored the cells source,cells culture,in vitro induction and differentiation,identification of pancreatic stem cells.Since the research of pancreatic stem cells is still in the early phase,there are lots of different methods and viewpoints,that is,no accepted strategy of culture,induction and idectification exist.In addition,since in vitro conditions can't mimic in vivo microenviroment that can make stem cells differentiated,the really mature islets cells can't be gotten in vitro and can't completely reverse the hyperglycemia.Therefore,we design this study in order to set up a simple and effective culture and identified protocol.Due to spikes,nutrient substance and factors exsiting in vivo microenviroment,we try to look for the best niche for pancreatic stem cells to differentiate,that is,a niche can make pancreatic stem cells to play an optimal role in the treatment of diabetes. Objective(1)To explore the methods of isolation,culture,identification and in vitro differentiation of pancreatic stem cells from newborn Wistar rats.(2)To explore the therapeutic efficacy of pancreatic stem cells with different differentiated status(such as undifferentiated stem cells and differentiated cells under the in vitro induction conditions)transplantation by two pathways,that is,portal vein and beneath renal capsule for the type 1 diabetes mellitus.Methods(1)The pancreatic tissue was removed form neonatal Wistar rats,digested into fragments by collagenase,then transferred into the culture flasks pretreated with collagen that contained RPMI1640 high glucose media(added 33.3mM Glu,10% FBS,20ng/ml EGF,and 20ng/ml bFGF),where pancreatic tissue was parimarily cultured and serially subcultured.The stem cells were induced and differentiated with RPMI1640 low glucose media without serum(reduced concentration of Glu to 11.1 mM,removed EGF,bFGF and FBS;and then added 10mM niacinamide and 71.5μMβ-mercaptoethanol).Ultrastructure of these cells was observed by scanning and transmission microscope electron.The expression of the antigens such as insulin, glucagon,PDX-1,CK-19 and Ngn3 was detected with immunofluorenscence.The ratio ofβcells was detected with DTZ staining.The insulin secreted by cells was determined with RI.PCR was performed to detecte the expression of PDX-1 mRNA, CK-19 mRNA,Ngn3 mRNA and insulin mRNA.(2)50 Wistar rats were injected through caudal vein with streptozotocin.The successful diabetes models can be concluded when random blood glucose was continuously more than 16.7 mM during one week.Then all rats were randomly divided into five groups:A group(n=5,as control,PBS was injected via portal vein), B group(n=10,undifferentiated pancreatic stem cells were transplanted via portal vein),C group(n=10,differentiated cells were transplanted via portal vein),D group (n=10,undifferentiated cells were transplanted beneath renal capsule)and E group (n=10,differentiated cells were transplanted beneath renal capsule).The random blood glucose,weight,plasma insulin and introperitoneal glucose tolerance test was detected after transplantation.All the rats were sacrificed respectively on 14 and 30 days postoperatively.The liver specimen of A,B and C group,and the kidney specimen of D and E group were removed to make pathological sections and immunohistofluorescence,measure the diameter of islet-like structures,and observe morphous and insulin expression.Results(1)Collagenase digestion in combination with high glucose media can effectively isolate and get purified pancreatic ductal epithelial cells.The cultured cells had the ability of high self-duplication,which show the characteristics of undifferentiated cells and expressed PDX-1 and CK-19 simultaneously.With the passage increased, some of these cells expressed Ngn3,but didn't express the antigens,like insulin and glucagon.Both DTZ staining and insulin secretion tests didn't show insulin-generated cells.Onec the cells differentiated,some cells can express insulin and glucagon,and ratio of DTZ-positive cells increased to 25±6%,which had an increased ability of insulin respondence to glucose with a range of 10-30 mM.(2)48 out of 50 Wistar rats were successfully made to diabetes models and the successful rate was 96%.Between 6 and 18 days after transplantation,the random blood glucose of B group has significant difference compared to that of control group, which once reduced to the normal range.C and E group's blood glucose had the similar tendency.Compared respectively to the control group,the difference of blood glucose on 4th day after transplantation was significant,but the time duration lasted too short.During the experiment,the blood glucose in both groups didn't reduce to the normal range.Compared to the control group,the blood glucose of D group didn't have statistical significance during the process.Considering the introperitoneal glucose tolerance test,the grafts in B group have better antihyperglycemic effect 13 day after transplantation.The undifferentiated pancreatic stem cells can be induced into the larger islet-like structures in liver,the diameters of which ranged from 150-200μm or so.The intensity of insulin-generated cells was strong.In both C and E group,small islet-like structures were usually 50μm in diameter,and the intensity was weak.Immunohistofluorescence examination showed BrdU positive cells of B group that surrounded the islet-like structures were more than those of C and E group.Conclusions(1)In this study,collagenase digestion was used to get the cells with ability to self-duplicate and potency to differentiate into insulin-generated cells,which can be proved to originate from ductal epithelium through many different kinds of experimental techniques.The condition of low glucose media contributes to the maturity of pancreatic stem cells.(2)The differentiated pancreatic cells under in vitro induced condition can further become mature and differrentiate many small islet-like structures.While in the circumstances of in vivo,i.e.,the liver,undifferentiated pancreatic stem cells can differentiate into more full-grown large islet-like structures,which can transiently totally reverse the high blood glucose of Wistar rats.The potency of pancreatic stem cells to differentiate into endocrine and excrocrine cells might contribute to the further maturity of insulin-generated cells. |
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