【Abstract】 Objective To explore good methods for isolation and purification of rat islets. Methods The isletswere isolated from male SD rat pancreata by a collagenase perfusion method and purified by a modified method: added 4 kinds of Euro-Ficoll of different densities (F1: D=1.132, F2: D=1.108, F4: D=1.069, F5: D=1.023), discontinuous density gradient centrifuge the tube at 2 000 r/min for 20 minutes at 4℃ , then the islets between F1 and F2 were collected. The purity of islets was assessed by dithizone staining with islets counted and scored for size. Islets viabil ity was assessed by fluorescin diacetate / propidium iodide. The function of purified islets was judged by the test of insul in release and islets transplantation. Results After an improved method for optimized isolation and purification, (920±122) IEQ purified islets were obtained from one rat. Both the purity and viabil ity of islets were over 90%. The amount of insul in secretion was (18.25±0.32) mU/L and (36.70±3.57) mU/Lat 2.2 mmol/ L and 22.2 mmol/L concentration of glucose respectively, there was significant difference between the two phases(P lt; 0.05). The insul in release index was 2.01±0.15. Under 1 000 IEQ islets transplantation, the normal glucose level could beremained in diabetic rats. Conclusion High purity and high viabil ity islet cells can be got through improved collagenase perfusion and centrifugation on gradients method.
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 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.
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.
Objective To introduce a new method of tissue engineering research by transplanting vessels to tissue engineering chamber (vascularized tissue engineering chamber) in vivo, and to review the progress of research in vascularized tissue engineering chamber. Methods The l iterature concerning all kinds of tissue engineering research in chamber was reviewed, analysed, and summarized. Results The use of vascularized tissue engineering chamber allowed generation of vascularized adipose tissue, cardiac tissue, and so on. The most common tissue engineering chamber models were arterio-venous loop model and inferior epigastric artery model. Conclusion The method of tissue engineering research by using vascularized tissue engineering chamber has a potential cl inical value and provides a promising future.
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 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 review the common methods of isolation and purification of porcine islets and research progress. Methods Domestic and abroad literature concerning the isolation and purification of porcine islets was reviewed and analyzed thoroughly. Results The efficacy of the isolation and purification depends on the selection of donor, the procurement and cryopreservation of high-quality donor pancreas, and the selection and improvement of the operation. Conclusion The shortage of transplanted islets could be resolved by the establishment of standardized and optimal process, which may also promote the development of porcine islet xenograft.
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 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.