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Objective To explore the function and survival of islet grafts during co-transplantation with adipose mesenchymal stem cells (AMSCs) in diabetic mice .Methods After human AMSCs and islet cells were isolated ,purified and then subcutaneously co-transplanted into nude mice with diabetes mellitus . Four groups of AMSCs + islet co-transplantation , islet transplantation alone ,phosphate buffered solution (PBS) and normal control mice were designated . Islet cell activity and apoptosis/revascularization degree of islet grafts were observed by immunohistochemical double staining of insulin ,factor associated suicide (Fas) and CD31 antibody . The blood glucose and serum insulin levels of mice and the survival time of islet grafts were compared . Results The blood glucose and serum insulin levels of diabetic mice analyzed by multivariate analysis in AMSCs + islet co-transplantation group were better than those in islet transplantation alone group (P< 0 .05 ) . The mean survival time (MST ) of islet grafts was longer in AMSCs + islet co-transplantation group than that in islet transplantation alone group [(81 .33 ± 7 .58) vs .(58 .17 ± 6 .91) days] (P<0 .05) .At Day 7 post-transplantation ,insulin staining intensity of islet grafts was higher in AMSCs + islet co-transplantation group than that in islet transplantation alone group while Fas staining intensity of islet grafts was lower .And mean microvascular density (MVD) of islet grafts per square millimeter was higher in AMSCs + islet co-transplantation group than that in islet transplantation alone group [(21 .8 ± 5 .6 ) vs . (14 .6 ± 4 .1 )] ( P< 0 .05 ) .Conclusions Co-transplantation with AMSCs may improve the function of islet grafts ,prolong its survival and promote its revascularization .
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Objective To investigate the effect of Notch signaling during bone marrow mesenchymal stem cells (BMSC) differentiating into islet in vitro.Methods The specific inhibitor of γ-secretase DAPT was used to inhibit the Notch signaling pathway.Mter induction,DTZ staining,indirect immunofluorescence staining,RT-PCR and Westenn blotting were used to detect the expression of insulin,glucagon,Pdx-1 and Ngn3.Results (1) Identification of BMSCs:Indirect immunofluorescence staining showed that BMSCs could express CD59 and CD90,which both were makrers of mesenchymal stem cells.Besides,BMSCs could express nerve culluar markers such as NSE,GFAP,suggesting multi-directional differentiation.(2) The result of MTT showed DAPT could inhibit the cell proliferation in a time-dependent manner and a dose-dependent mannar.Besides,DAPT could inhibit the expression of target gene of Notch signal pathway in a time-dependent manner and a dosedependent mannar.After treated by 1,5,20 μmol DAPT,the expression of Hes1 had reached to 92.06％,71.40％ and 46.89％ of controls respectively,suggesting efficiency of inhibition on Notch reached 7.94％,28.6％ and 53.11％ respectively (all P＜ 0.05).(3) Indirect immunofluorescence staining showed the expression of pancreas-specific markers such as insulin and glucagon were much higher in DAPT treated BMSCs than that in controls,which was confirmed by RT-PCR and Western blotting analyses.The proportion of insulin-producing cells differentiated from DAPT treated BMSCs was (74.03 ± 3.96)％,which was higher than that from controls[(36.49 ± 3.24)％,P＜ 0.05].(4)Furthermore,RT-PCR and Western blotting analysis showed that the expressions of Pdx-1 and Ngn3 were earlier than that of insulin and glucagon,and the expressions of Pdx-1 and Ngn3 were higher in DAPT treated BMSCs than that in controls.Conclusions Notch signaling pathway plays a role in the differentiation of BMSCs into islet in vitro.Pharmacological interference with Notch signaling pathway may provide a novel method to obtain islet for therapeutic use.
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Allo-islet transplantation is believed to be a promising treatment for normalizing blood glucose levels without hypoglycemic episodes in patients with type 1 diabetes mellitus (T1DM). In 2000, a pioneering study by the Edmonton group showed that allo-islet transplantation could achieve insulin independence for at least 1 year post-transplantation in all seven consecutive patients. This breakthrough study excited numerous researchers, clinicians, and patients. Although longer follow-up studies did not have the same success as the first study, substantial efforts to establish successful islet transplantation have been made in the last decade. Several leading centers of islet transplantation have reported success rates of nearly 50% insulin independence at 5 years post-transplantation. However, recent advancements in transplant outcomes are limited to only a few centers and select patients; thus, we are still confronted with numerous hurdles against long-term successful islet transplantation. Herein, we review the recent advances and challenges for allo-islet transplantation to be accepted as a standard therapy for patients with T1DM.
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Humans , Blood Glucose , Diabetes Mellitus , Diabetes Mellitus, Type 1 , Follow-Up Studies , Insulin , Islets of Langerhans TransplantationABSTRACT
Objective To observe the effects of intensive insulin treatment on islet β cell apoptosis associated protein bcl-2 and bax in type 2 diabetic rats.Methods 36 Wistar rats were randomly divided into two groups : normal control group and high fat diet group.Rats in normal control group fed by basical feedstuff.Rats in high fat diet group fed by high fat and basical feedstuff.After 10 days,rats in high fat group were injected with STZ.After 3 days,rats in high fat group were randomly divided into two groups:diabetes control group and insulin treatment group.The course of treatment was 4 weeks.After 10 days by fat milk intragastfic administration, after 3 days of STZ injection and after 4 weeks treatment, each index was measured.After experiment, pancreatic tissue bel-2 and bax were detected through immunohistocbemical method.Results After 4 weeks intensive insulin treatment,the bcl-2 was significantly increased at(6.20 ± 2.05 )% in insulin treatment group than diabetes control group.The bax was significantly decreased at ( 2.68 ± 1.04 ) % in insulin treatment group than diabetes control group ( P < 0.05 ).Conclusion The method of insulin intensive treatment could increase islet βcell bcl-2 and decrease bax in type2 diabetic rots, Insulin intensive treatment could decrease islet β cell apoptosis.
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Objective To analyze the protection supplied by HSCs in vivo after islet cells homotransplantation. Method Diabetic mice were randomly divided into three groups as diabetic group,islet-transplant group and co-transplant group. 300 islets alone or mixed with HSCs were transplanted under the capsule of the kidney of the diabetic recipients respectively. Blood glucose and the length of normal blood glucose level were recorded and we collected the blood and tissue of islet graft 7 days after transplantation in each group. Blood concentration of TGF-β, TNF-α, IL-1β and IFN-γ were detected by ELISA. The infiltration of lymphocytes was observed by HE staining and immunohistochemieal examination.Result Co-transplant group had a prolonged islet allograft survival for (23.75 ± 8. 96) days, compared with the islet alone of (11.9 ± 6. 92) days. Blood concentration of TGF-β in co-transplant group was (2292.31 ± 5.87) pg/ml, significantly higher than those in simple transplant group (1246.55 ±38.91) pg/ml(P <0.05), there was no differentence in the two groups for TNF-α,IL-1β and IFN-γ.Conclusion HSCs may prolong the islet graft survival by expressing higher level of TGF-β and form a capsule around the graft.
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Objective To investigate the therapeutic effect of frozen-thawed murine islets which were transplanted into diabetic rats after cultured with hyperbaric oxygenated rotary cell culture system (HORCCS). Methods The purified rat islets were divided into two groups: A. In vitro experiment groups (IvEG) : The rat islets in each subgroup were cultured in HORCCS or common medium for 30 days, then evaluated for the intracellular DNA and insulin contents of islets, and the viability and insulin secreting level of islets. B. Islet transplantation experimental groups (TxEG) : The frozen-thawed islets were cultured in HORCCS or common medium for 7 days, and then transplanted into the recipients. We observed the blood glucose level (BGL) and insulin secreting level in the recipients as well as the uhrastructure change of islets in TxEG. Results The viability and insulin secreting level of islets cultured with HORCCS at 14th day were much higher than those cultured with common medium (P <0.05). The blood glucose level in recipients transplanted with islets cultured with HORCCS recovered to normal value at the 2nd week and lasted for 8 weeks. All these recipients maintained the normal glucose tolerance curve. Electronic microscopy found microchannel outlets on the surface of the frozen-thawed islets cultured with HORCCS. Conclusions Frozen-thawed islets cultured with HORCCS could establish nutrient transmission microchannels, which were not only capable of oxygen and nutrients transmission, but also improving cryopreservation solution to diffuse inside the islet cells evenly and uniformly. So this method not only lessens islet damage from cryopreservation, but also improves the effect of transplantation.
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Objective To investigate the inhibitory effects of Argatroban on the instant bloodmediated inflammatory reaction(IBMIR)after islet transplantation.Methods Rat islets were isolated and purified rat islets,and were divided into blank control group,control group and experimental group.In the control group,the blood and the islets were directly mixed,and in the experimental group the Argatroban was added to the mixture based on the control group.while the blank control group was added with blood alone without the islets.Each group was reacted at 37℃for 60min,and then the content was filtered through trap valve of 70 μm.The residual thrombus and tissues were filtered by the trap valve in both the experimental and control groups,detected by the thinprep cytologic test(TCT),and the filtrate received blood routine test,and the function of islet was determined using insulin releasing test.Results The number of blood platelets,white blood cells,mononuclear cells,and lymphocytes percentage in the experimental group were significantly higher than in the control group after 60 min(P＜0.05).Under the environment of the high and low concentrations of glucose,the insulin release in experimental group was significantly increased as compared with the control group,and the insulin release index of former was 2.25±0.18,significantly higher than that of the latter 3.36±0.18(P＜0.05).The residual thrombus and tissues had few islet cells in the control group,the structure was damaged seriously,the capsule was not intact,and there were a large mumber of micro-thrombosis around the islets formed by red blood cells.But there were a large number of islet cells in the experimental group.the structure was intact in a mass,and no obvious micro-thrombosis around the islets was found.Conclusion Argatroban can effectively inhibit IBMIR in vitro,and alleviate the damage to the islet cells.
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Purpose of review The enthusiasm generated by the results of the Edmonton protocol of islet transplantation is inciting a great number of institutions to start such programs. However, the procedure of islet isolation and purification is costly, complex and technically challenging. In order to share costs and to avoid facing the steep learning curve of the procedure, many centers interested in islet transplantation have looked into collaborating with experienced groups serving as core islet isolation facilities. Recent findings The proof of principle that remote islet processing and shipment could be successfully implemented with obtainng the Portland/Minneapolis, Huddinge/Giessen and Houston/Miami partnerships. Moreover, in order to increase both the donor pool and the number of patients gaining access to islet transplantation, multicenter networks,such as the Swiss-French GRAGIL consortium and the 4-country Nordic Network in Scandinavia have been built. The GRAGIL group has been fully operational since 1999, allowing the transplantation of 27 islet preparations processed in Geneva, Switzerland into 20 recipients in France over the course of 4.5 years.Organizational issues in the design of such networks are discussed based on the example of the GRAGIL experience. Summary The feasibility and the efficiency of islet transplantation in multicenter networks have been demonstrated. This strategy allows to increase the donor pool and the accessibility to islet transplantation in an extended population area. (J Intervent Radiol, 2006, 15:626-631 )
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Objective To observe the protective effect of Ulinastatin in rat islet isolation and advance an improved method in rat pancreas islet isolation.Methods Rat pancreas was perfused with Hanks solution containing collagenase. The rats were divided into 3 group according the Ulinastatin concentrations in collagense: low-concentration Ulinastatin ( 2500 U/ml) group, high-concentration Ulinastatin ( 5000 U/ml) and control group (no Ulinastatin). The isolated pancreases were compared among the groups. Results The shape and number of the isolated pancreases in low-concentration Ulinastatin and high-concentration Ulinastatin groups were superior to those in control group (P
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Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microgram/microliter cDNA and 25 microliter effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.
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Animals , Male , Mice , Body Weight , Cell Survival , Diabetes Mellitus, Experimental/complications , Disease Models, Animal , Glucose/pharmacology , Glucose Tolerance Test , Hyperglycemia/complications , Insulin/metabolism , Islets of Langerhans/blood supply , Islets of Langerhans Transplantation , Liposomes/administration & dosage , Mice, Inbred BALB C , Neovascularization, Physiologic , RNA, Messenger/genetics , Streptozocin , Transfection , Vascular Endothelial Growth Factors/biosynthesisABSTRACT
Heterotopic pancreas can be defined as the occurrence of pancreatic tissue at aberrant anatomic sites that lack vascular, neural and anatomic community with the pancreas. A minority of heterotopic pancreas elicit clinical signs or symptoms that can vary according to its location and size, and the involvement of the overlying mucosa. We report here on a case of heterotopic pancreas that was removed by pancreatoduodenectomy under the erroneous diagnosis of distal common bile duct cancer.
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Ampulla of Vater , Common Bile Duct , Diagnosis , Jaundice , Mucous Membrane , Pancreas , PancreaticoduodenectomyABSTRACT
To examine the localization pattern of phospholipase D2 (PLD2) in the pancreatic islet (the islet of Langerhans) depending on species, we conducted a morphological experiment in the rat and guinea pig. Since individual islets display a typical topography with a central core of B cell mass and a peripheral boundary of A, D, and PP cells, double immunofluorescent staining with a panel of antibodies was performed to identify PLD2-immunoreactive cells in the islets PLD2 immunoreactivity was mainly present in A and PP cells of the rat pancreatic islets. And yet, in the guinea pig, PLD2 immunoreactivity was exclusively localized in A cells, and not in PP cells. These findings suggest a possibility that PLD2 is mainly located in A cells of rodent pancreatic islets, and that the existence of PLD2 in PP cells is not universal in all species. Based on these results, it is suggested that PLD2 may play a significant role in the function of A and/or PP cells via a PLD-mediated signaling pathway.
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Animals , Rats , Antibodies , Guinea Pigs , Guinea , Islets of Langerhans , Phospholipases , RodentiaABSTRACT
Objective: To investigate the possible effects of Pancreatic duodenal homeobox-1((Pdx-1))expression in transdifferentiation of bone marrow mesenchymal stem cells(MSC) in vitro. Methods: The eukaryotic expression vector containing Pdx-1 was constructed.After such vector was transfected into MSC using Superfect,G418 was used to select the positive cells.Then both(Pdx-1~(+)MSC)and((Pdx-1~-MSC)) were induced to transdifferentiate in vitro.The expressions of insulin were analyzed by immunohistochemistry staining. Results: Restricted enzyme analysis and sequencing showed that interest gene segment was consistent with that in Gen Bank,Recombination vector was effectively transformed into MSC demonstrated by fluorescence microscope;insulin-producing cells from Pdx-1~(+)MSC were higher than that from Pdx-1~(-)MSC[(28.23?2.56)% and(7.08?2.69)%,respectively]. Conclusion:Pdx-1 can promote adult rat MSC to transdifferentiate into insulin-producing cells in vitro,and this approach might lead to a widespread cell replacement therapy for type I diabetes.
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Objective To explore whether sodium and magnesium saline of fructose-1,6-diphosphate(Na +-Mg 2+-FDP)protects B cells in islet of Langerhans injured by streptozotocin(STZ).Methods Healthy adult male Wistar rats were randomly divided into five groups:STZ group(receiving once 2% STZ in citric acid solution at dose of 60 mg/kg),blank control group(receiving citric acid solution),FDP group(treated as STZ group,plus Na +-Mg 2+-FDP solution twice a day at total dose of 500 mg?kg-1?d-1 for 15 d),protected group(receiving Na +-Mg 2+-FDP solution at 500 mg/kg first,then 2% STZ in citric acid solution at dose of 60 mg/kg once,Na +-Mg 2+-FDP solution twice a day at total dose of 500 mg?kg-1?d-1 for 15 d),treated group(48 h after receiving 2% STZ in citric acid solution at dose of 60 mg/kg,starting Na +-Mg 2+-FDP solution twice a day at total dose of 500 mg?kg-1?d-1 for 15 d).On the 15 th day after experiment starting,all rats were killed and the end of pancreatic gland was prepared for following experiments.Blood glucose by using glucose oxidase kit and serum insulin by radioimmunoassay was determined.The number of positive islets of which A,B and D cells expressed glucagons,insulin and somatostatin was counted by S-P immunohistochemistry.The morphology of islet of Langerhans stained by HE was observed under optical microscope.Statistical significance was determined by q test and ? 2 test,the related coefficient was determined by rectilinear correlation analysis(?=0.05).Results The blood glucose dropped and serum insulin rose in protected group,but the glucose in treated group and STZ group maintained at high level,and serum insulin maintained at low level.The blood glucose and insulin of the protected group showed significant differences with that of the treated group and STZ group respectively(P0.05).However,the expressions of insulin,gluca-gons and somatostatin were close to normal level in protected group,which were of statistical significance with that of STZ group and treated group(P
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Objective To explore the isolation, culture of nestin positive cells of the pancreas in newborn rats. Methods The whole pancreas of neonatal rats were digested with collagenase, followed by incubation under pH 7. 6 serum RPMI 1640 for 24 - 36 h and then under pH 7. 4 serum free RPMI 1640 (bFGF.EGF 20 ng/ml.1% N2) for 18-24 d. The expression of insulin.glucagon and nestin were detected by immunofluorescence technique. Nestin.CK19 were detected by RT-PCR. Resides Cells attached to the surface of plates after 24 h incubation under pH 7. 6 condition, and islet-like masses were obtained after 18 -24 d incubation. A monolayer of cells grew out after 24 h of passage of islet-like masses. Nestin positive cells was detected after 24 - 36 h incubation, with no expression of insulin and glucagon. Positive cells of insulin and glucagon were detected in islet-like masses after 24 h passage. Nestin positive cells were detected by RT-PCR in islet-like masses after 24 h passage, but no CK19. Conclusion Insulin and glucagon were expressed in islet-like masses after passage. Nestin positive cells in the pancreas of neonatal rats possessed character of islet stem cells.
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Objective To identify the effect of peptide nucleic acid of CC chemokine receptor 5 on acute rejection of islet allograft.Methods Mice islet transplant models were used to test the effect of PNA CCR5 by targeting CCR5 in acute allograft rejection.In vitro T cell proliferative responses were assessed by mixed lymphocyte response(MLR).RT-PCR and Western blot were used to detect the expression of mRNA and protein.Results PNA CCR5-treated recipients demonstrated statistically significant prolongation(12.00?1.75)d in functional allograft survival when compared with saline(6.50?0.58)d or PNA mismatch-treated recipients(6.50?0.50)d.The CCR5 mRNA expression level of PNA CCR5,control,and PNA mismatch treatment recipients at day 7 posttransplant was 0.56?0.05,1.68?0.07 and 1.80?0.14,respectively.The data showed that CCR5 protein was significantly down-regulated in PNA CCR5 treatment allografts compared with saline and PNA mismatch treatment allografts(P
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Objective To assess the effect of fetal islet transplantation for the treatment of Type Ⅰ diabetes. Methods The pancreatic islets from human aborted embryos were cultured and implanted into the greater omentum and omental bursa of 26 patients with type Ⅰ diabetes. The function of transplanted islets was evaluated. Results After transplantation, the exogenous insulin requirement significantly decreased (P
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Both ?-cell dysfunction and insulin resistance contribute to the development of type 2 diabetes, and ?-cell function, especially early insulin secretion, is important in maintaining normal glucose tolerance. This review focuses on the ciritical role of ?-cell dysfunction in the pathogenesis of type 2 diabetes.
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Objective To investigate a new method of a long term culture for rat islets in vitro. Methods Rat islets marked by green fluorescent protein (GFP) were cocultured with Sertoli cell for 20 weeks. Histological studies were performed on islet group and coculture group in 1w, 3w, 10w, 15w, 20w of culture by light, fluorescent and electron microscopy. Insulin released was measured by radioimmunoassay. Results In islet group, islet viability and the number of insulin positive cells were significantly decreased after 3w of culture, cumulative quantities of insulin for 24 hours and the stimulation index also fell rapidly under this condition, meanwhile the ultrastructure of islets was destroyed. However, under coculture condition, culture time of islets was prolonged in vitro, islet viability and the number of insulin positive cells were significantly increased, cumulative quantities of insulin for 24 hours and the stimulation index maintained at high level, and the ultrastructure of islets remained normal even after 20w of culture. Conclusion Coculture of rat islets with Sertoli cells may promote islet growth and prolong culture time, and it is a new method of a long term culture of islets in vitro.