Objective To study the advances in microcirculation after islets of Langerhans transplantation (ILT). Methods The literature in the recent years on the study of the relationship between ILT and microcirculation was reviewed. Results The process of angiogenesis and revascularization of the islet grafts was in progress within 1 week after transplantation, and was completed within 10-14 days after transplantation, exhibiting a microangioarchitecture similar to pancreatic islets in situ. The sequence of vascular intraislet cellular perfusion was from β cells outward to α-and δ-cell cortex, with the majority of α cells perfused before the majority of δ cells. Freely transplanted islet grafts were revascularized from the hostderived microvascular bed. The interstitial pressure in the islet transplants was markedly lower than the capillary pressure. There were clearly differences in microcirculation between syngeneic and xenogeneic islet grafts. The phenomena of microcirculation failure were observed in xenografts. The influential factors of microcirculation after ILT were ①culture temperature of isolated islets, ②cultured time and cryopreserved method of islets, ③blood glucose, ④immunosuppressive agents, ⑤angiogenesis factors. Conclusion Microvascularization of freely islet grafts is one of the essential requirements for successful engraftment, guaranteeing sufficient nutritional blood supply to the tissue and establishing blood drainage for adequate liberation of the endocrine hormones. Through the studies of the microcirculation after ILT, it is helpful to recognize the mechanism of the survival of islet grafts.
Objective To investigate differential points of clinical symptoms and pathology of solid-pseudopapillary tumor of the pancreas (SPTP) and islet cell tumor (ICT). Methods Fifteen cases of SPTP and twelve cases of ICT were studied in this retrospective research. Clinical symptom, pathologic feature and computed tomography (CT) image of patients with both tumors were analyzed, and the imaging features were compared with pathological results. Results The mean age of SPTP patients was 22.4 year-old. Twelve patients with SPTP presented a palpable abdominal mass as the initial symptom. It was observed that the tumor cells were located in a pseudopapillary pattern with a fibro-vascular core histologically. On the CT images, a mixture of solid and cystic structures could be seen in all the tumors. After taking enhanced CT scan, the solid portion was slightly enhanced in the arterial phase and the contrast intensity increased in the portal venous phase. On the other hand, the mean age of ICT patients was 39.3 year-old. The major symptom was due to the function of islet cell tumor, which was typical in 8 patients, presenting as Whipple triad. Histologically, cells demonstrated in trabecular, massive, acinar or solid patterns, and the blood supply of the tumor was abundant. On the CT images, most small tumors were difficulty to be detected. ICT could be markedly enhanced in the arterial phase and slightly enhanced in the portal venous phase on post-contrast CT scan. Conclusion Clinical symptom, pathologic feature and CT scanning are helpful to differentiate SPTP from ICT.
ObjectiveTo investigate the significant effect of costimulatory pathway B7CD28/CTLA4 on the islets of Langerhans transplantation. MethodsThe literatures were reviewed to summarize the molecular structure and functions of the pathway and the related animal experiments.ResultsThe costimulatory pathway B7CD28/CTLA4 was one of the signaling pathways of T cells activation and proliferation. If the costimulatory signals were absent, Tlymphocyte would be induced to the clonalanegy. Through blocking the costimulatory pathway mediated by CD28, CTLA4Ig prolonged to the islets of Langerhans survival in recipients. ConclusionBy the studies of the costimulatory pathway, it is helpful to understand the immune mechanism of the survival of islet grafts.
Objective The purity and activity of islets will greatly affect the outcome of xenotransplantation therapy of type 1 diabetes mell itus. To set up an improved method of the isolation and purification of rat islets, which can obtain highpurity,high-yield, and high-viabil ity islets. Methods Ten healthy and adult male SD rats, weighing 250-300 g were used asorgan donors. Collagenase V was perfused into pancreas via pancreatic duct. Pancreas was digested with collagenase in water bath at 38℃ about 15 minutes, islet purification was performed using two techniques: with Ficoll 400 density gradient (group A), and Ficoll-Paque™ PLUS (group B). Dithizone (DTZ) was util ized for identifying islets, counting islets equivalent quantity (IEQ) and islets’ purity. Trypan blue staining was used to detect the viabil ity of islets. Islets of group B was encapsulated with alginate/poly-L-lysine/alginate (APA). Islets function of microencapsulated and nonmicroencapsulated was evaluated by the insul in release test. Results DTZ staining showed that islets shape were round, ell ipse and irregular with a clear edge and a diameter range of 50-300 μm. The IEQ values were 338.04 ± 76.61 and 834.80 ± 54.00 in groups A and B, respectively, showing significant difference (P lt; 0.05). The purities were 88.31% ± 2.67% and 95.63% ± 1.96% in groups A and B, respectively, showing no significant difference (P gt; 0.05). The activities of islets were 67.40% ± 5.15% and 86.05% ± 2.52% in groups A and B, showing significant difference (P lt; 0.05). Islet APA microcapsules had round shape, unified size, and its diameter was between 1.5 and 2.0 mm. Each microcapsule was encapsulated of 1 to 3 islets. The result of insul in release assay was that the concentrations of insul in secretion with islets of microencapsulated and nonmicroencapsulated were (5.53 ± 1.64) ng/ mL and (4.76 ± 0.26) ng/mL in low glucose, and its concentrations of insul in secretion in high glucose were (11.95 ± 2.07) ng/ mL and (14.34 ± 3.18) ng/mL. Stimulated insul in secretion in high glucose was 2 times more than that in low glucose (P lt; 0.05), but there was no significant difference (P gt; 0.05) in the stimulation index between group A (2.16 ± 0.30) and group B (3.01 ± 0.59). Conclusion The method of islets isolation and purification using Ficoll-Paque™ PLUS own the virtues of more convenient, high islet yield, and high islet purity. Both microencapsulated and nonmicroencapsulated islets show high-viabil ity while culture in vitro.
Diabetes is characterised by hyperglycaemia resulted as the relative or absolute insulin deficiency which is closely related to islet beta cell failure. Apoptosis is the core mechanism of beta cell failure according to the studies on human islet. However, apoptosis can’t fully explain the loss of beta cell mass in the process of type 2 diabetes or the protective effect of early intervention. Recently, some other possible mechanisms of beta cell dysfunction have been proposed and dedifferentiation of beta cell draws extensive attention. Evidences of beta cell dedifferentiation in type 2 diabetes patients and animal models outlined and the transcription factors which determine beta cells of identity during this procedure are discussed in this review.
Objective To investigate the effect of constitutively active Akt1 gene on rat engrafted islets in apoptosis and revascularization, and to explore potential method of gene therapy in the islet transplantation. Methods Rat islet which was transfected constitutively actived Akt1 gene via adenovirus vector using MOI=500. Thirty-six streptozotocin induced diabetic Wistar rats were divided into 3 groups complete randomly: Adv-CA-Akt1 group, Adv-LacZ group and simple transplantation group. Blood glucose and insulin were determined after operation. TUNEL was used to detect the apoptotic islet cells. HE and immunohistochemical staining of insulin were used to evaluate the histology of the islet grafts. The microvessel density (MVD) was determined by CD31 immunohistochemical staining. Results The fasting glucose level in Adv-CA-Akt1 group restored to normal 2 days after transplantation. However, in Adv-LacZ group and simple transplantation group, it reduced but still kept being hyperglycemia. And the serum insulin level was higher than other two groups ( P ＜ 0.05). Compared to simple transplantation group and Adv-LacZ group, apoptotic rate decreased 25% in Adv-CA-Akt1 group, a large number of islet grafts were seen under the capsule of the kidney, which were positively stained by insulin antibody. In the other two groups, the islet groups mass were lighter, and few positively stained by insulin antibody. MVD showed lighter positive endothelial cells stained by CD31 antibody in the other two groups than Adv-CA-Akt1 group ( P ＜ 0.05). Conclusion Constitutively activate Akt1 gene can prolong graft survival during early posttransplant period, and can accelerate the revascularization of islet grafts effectively.
ObjectiveTo explore the endothelial cells from human peripheral blood and islet of rat co-transplantation under the renal capsule of diabetic nude mice to improve the survival and function of transplanted islet. MethodsThe endothelial cells from human peripheral blood(5×105)and freshly isolated rat islet cells were co-transplanted under the renal capsule of diabetic nude mice model, then the fasting blood glucose, body weight, peripheral blood C-peptide level, and intraperitoneal glucose tolerance test(IPGTT) were measured to evaluated the islet graft survival and function. ResultsCompared with the control group, the fasting blood glucose level significantly decreased(P < 0.01), peripheral blood C-peptide level rised(P < 0.01), and body weight increased(P < 0.01) of receptor nude mice in experience group, the IPGTT also improved. ConclusionThe endothelial cells from human peripheral blood and islet of rat co-transplan-tation can obviously improve the survival and function of transplanted islet of nude mice.
Objective To summarize the research progress on the source and selection of donor cells in the field of islet replacement therapy for diabetes mellitus. Methods Domestic and abroad literature concerning islet replacement therapy for diabetes mellitus, as well as donor source and donor selection was reviewed and analyzed thoroughly. Results The shortage of donor supply is still a major obstacle for the widely clinical application of pancreatic islet transplantation (PIT). Currently, in addition to the progress on the allogeneic/autologous donor islet supply, some remarkable achievements have been also attained in the application of xenogeneic islet (from pig donor), as well as islet like cells derived from stem cells and islet cell line, potentially enlarging the source of implantable cells. Conclusion Adequate and suitable donor cell supply is an essential prerequisite for widely clinical application of PIT therapy for type 1 diabetes mellitus (T1DM). Further perfection of organ donation system, together with development of immune-tolerance induction, gene and bioengineering technology etc. will possibly solve the problem of donor cell shortage and provide a basis for clinical application of cellular replacement therapy for T1DM.
Objective To study the protective effects of bone marrow mesenchymal stem cells (BMSCs) of rhesus monkeys on porcine islets from hypoxia/reoxygenation (H/R)-induced injury. Methods BMSCs were isolated and cultured from the marrow of 5 adult rhesus monkeys (weighing, 6-10 kg) by adherent monocytes. Islets were isolated and purified from the pancreas of 5 neonatal porcine (3-5 days old) by collagenase V digestion method, and were cultured with or without BMSCs, and exposed to hypoxia (1%O2) for 12 hours and reoxygenation for 24 or 48 hours, respectively. The experiment was divided into 4 groups: normal islet group (group A), normal islet + BMSCs group (Group B), H/R islet group (group C), and H/R islet + BMSCs group (group D). The survival rate of islets was calculated by fluorescein diacetate/propidium iodide (PI) staining. The viability of the islet cells was detected by cell counting kit 8. Apoptotic rate of islet cells was tested using Annexin V-FITC/PI labeling and flow cytometry. The stimulation index (SI) of islet function was analyzed by glucose-stimulated insulin secretion assay. Results The islet cell cluster of group C was more dispersed than that of groups A and B, and group C had more death cells; and the islet cell cluster of group D was more complete and the survival rate was higher than those of group C. The survival rate of islet was 90.2% ± 9.1%, 88.3% ± 5.9%, 52.3% ± 12.1%, and 71.4% ± 11.5% in groups A, B, C, and D respectively, it was significantly lower in groups C and D than in groups A and B (P lt; 0.05), but it was significantly higher in group D than in group C (P lt; 0.05). After coculture of BMSCs and islet at the ratio of 1 ∶ 10 and 1 ∶ 20 in group D, the viability of islet cells was significantly higher than that in group C (P lt; 0.05). The apoptotic rate was 27.1% ± 3.2%, 24.0% ± 1.0%, 64.3% ± 1.8%, and 46.2% ± 1.4% in groups A, B, C, and D respectively, it was significantly higher in groups C and D than that in groups A and B (P lt; 0.05), but it was significantly lower in group D than in group C (P lt; 0.05). There was no significant difference in SI between groups A and B at each time point (P gt; 0.05), but it was significantly lower in group C than in groups A and B (P lt; 0.05); and it was significantly higher in group D than in group C at 24 and 72 hours (P lt; 0.05). Conclusion BMSCs of rhesus monkeys can protect islet vitality and function from H/R-induced injury.
Objective To investigate the effect of inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine on pancreas islets cultured with cytokines TNF-α and IL-1β in rats. Methods Islets isolated from Wistar rats were purified and cultured. According to whether cytokines TNF-α, IL-1β and aminoguanidine were added into the medium respectively or not, islets were divided into 4 groups: cultured with islet only was taken as blank control group, cultured with TNF-α+IL-1β as cytokine group, cultured with aminoguanidine as aminoguanidine group, and cultured with TNF-α+IL-1β and aminoguanidine as aminoguanidine+cytokine group. NO level in culture medium and iNOS activity in islets tissue (Test Kit), apoptosis (TUNEL method) and viability of islets cell (acridine orange/ethidium bromide stain), and the function of islets (insulin release test) were measured. Results Compared with blank control group, the activity of iNOS in islet tissue and level of NO in culture medium increased, and the mass mortality and apoptosis appeared in islet cells, while insulin secretion decreased in cytokine group (P＜0.01). Compared with cytokine group, the activity of iNOS 〔（3.17±0.51） U/ml vs. （38.93±4.72） U/ml〕 and level of NO 〔（50.5±10.4） μmol/L vs. （313.0±35.4） μmol/L〕 decreased, the survival 〔（72.73±3.14）% vs. （57.07±5.07）%〕 increased and the apoptosis rate 〔（20.11±8.48）% vs. （41.17±6.87）%〕 decreased, the insulin secretion (secretion index: 3.50±0.27 vs. 1.96±0.19) improved； There were all significant differences in 2 groups (P＜0.01). Conclusion The iNOS inhibitor aminoguanidine could prevent the islet from the damage of iNOS/NO, alleviate the impairment of cytokines to islets, and ameliorate the survival and function of islets.