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 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.
ObjectiveGelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA)/chitosan oligosaccharide (COS) hydrogel was used to construct islet biomimetic microenvironment, and to explore the improvement effect of GelMA/HAMA/COS on islet activity and function under hypoxia. Methods Islets cultured on the tissue culture plate was set as the control group, on the GelMA/HAMA/COS hydrogel with COS concentrations of 0, 1, 5, 10, and 20 mg/mL respectively as the experimental groups. Scanning electron microscopy was used to observe the microscopic morphology, rheometer test to evaluate the gel-forming properties, contact angle to detect the hydrophilicity, and the biocompatibility was evaluated by the scaffold extract to L929 cells [using cell counting kit 8 (CCK-8) assay]. The islets were extracted from the pancreas of 8-week-old Sprague Dawley rats and the islet purity and function were identified by dithizone staining and glucose-stimulated insulin secretion (GSIS) assays, respectively. Islets were cultured under hypoxia (1%O2) for 24, 48, and 72 hours, respectively. Calcein-acetyl methyl/propidium iodide (Calcein-AM/PI) staining was used to evaluate the effect of hypoxia on islet viability. Islets were cultured in GelMA/HAMA/COS hydrogels with different COS concentrations for 48 hours, and the reactive oxygen species kits were used to evaluate the antagonism of COS against islet reactive oxygen species production under normoxia (20%O2) and hypoxia (1%O2) conditions. Calcein-AM/PI staining was used to evaluate the effect of COS on islet activity under hypoxia (1%O2) conditions. Islets were cultured in tissue culture plates (group A), GelMA/HAMA hydrogels (group B), and GelMA/HAMA/COS hydrogels (group C) for 48 hours, respectively. Immunofluorescence and GSIS assays were used to evaluate the effect of COS on islet activity under hypoxia (1%O2) conditions, respectively. Results GelMA/HAMA/COS hydrogel had a porous structure, the rheometer test showed that it had good gel-forming properties, and the contact angle test showed good hydrophilicity. CCK-8 assay showed that the hydrogel in each group had good biocompatibility. The isolated rat islets were almost round, with high islet purity and insulin secretion ability. Islets were treated with hypoxia for 24, 48, and 72 hours, Calcein-AM/PI staining showed that the number of dead cells gradually increased with time, which were significantly higher than those in the non-hypoxia-treated group (P<0.001). Reactive oxygen staining showed that GelMA/HAMA/COS hydrogels with different COS concentrations could antagonize the production of reactive oxygen under normal oxygen and hypoxia conditions, and this ability was positively correlated with COS concentration. Calcein-AM/PI staining indicated that GelMA/HAMA/COS hydrogels with different COS concentrations could improve islet viability under hypoxia conditions, and cell viability was positively correlated with COS concentration. Immunofluorescence staining showed that GelMA/HAMA/COS hydrogel could promote the expression of islet function-related genes under hypoxia conditions. GSIS assay results showed that the insulin secretion of islets in hypoxia condition of group C was significantly higher than that of groups B and C (P<0.05). Conclusion GelMA/HAMA/COS hydrogel has good biocompatibility, promotes islet survival and function by inhibiting reactive oxygen species, and is an ideal carrier for building islet biomimetic microenvironment for islet culture and transplantation.
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 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 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.
【Abstract】ObjectiveTo develop a method of adult porcine pancreatic islet isolation.MethodsThe tails of adult porcine pancreas were perfused through the pancreatic duct with 0.1% cold collagenase(type Ⅺ) and incubated at 38.5 ℃.The digested tissue was dispersed in 4 ℃ Hanks balanced salt solution(HBSS).The tissue suspensions were filtered through a 600 μm mesh.The residual tissue was resuspended in cold HBSS,and put in the Ricordi’s chamber and shaken for 5 minutes,then filtered again.The isolated islets were divided into three groups: control group(n=14),Pefabloc(trypsin inhibitor,n=8) group and FOY(trypsin inhibitor,n=5) group.The collagenase solution of the Pefabloc and FOY group was supplemented with 1.0 mmol/L Pefabloc and FOY respectively. ResultsThe islet yields of the Pefabloc group and FOY group 〔(11 848±3 530) islet/g pancreas and (14 496±3 693) islet/g pancreas〕 were significantly higher than that of the control group 〔(8 505±3 349) islet/g pancreas〕,P＜0.05.The activity of pancreatic protein enzyme in digestive fluid after digestion in control group was higher than the activity of pancreatic duct before injection and Pefabloc group(P＜0.01),which the control group, pancreatic duct before injection and Pefabloc group were (114.7±50.0) BAEEU,(4.0±1.8) BAEEU and (5.5±2.7) BAEEU,respectively.The pancreatic duct before injection and Pefabloc group showed no significant difference in statistics. In control group,when the harvest of islet was more than 8 000/g,the activity of pancreatic protoin enzyme was less than that with the harvest of islet below 8 000/g 〔(78.3±26.7) BAEEU vs (137.5±48.4) BAEEU,P＜0.05〕.Islet after purification in control group,Pefabloc group and FOY group showed good insulin secretion ability for different concentration of glucose.ConclusionA higher porcine pancreatic islet yield can be obtained by this method of pancreatic islet isolation and prophylactic administration of trypsin inhibitors consistently produce excellent islet yields.
Objective To study the effect of soybean trypsin inhibitor (STI) on the rat islets’ yield and function during the process of isolation and purification. Methods The rats were divided into experiment group and control group according to whether STI was put into the collagenase. STI (2.0 mg/ml) was put into the collagenase digestive juice of the experiment group and none to the control group. For both of two groups, islets were isolated by situ perfusion collagenase into the rat pancreas and they were purified by un-continuous gradient centrifugation with Ficoll-400. The quantities of the obtained rat islets before and after purification were recorded, and the morphosis and function of the purified rat islets were tested, then their vivo function were observed after islets plantation. Results After digest and before purification, there was no obvious deviation of the obtained islets quantity between two groups 〔(624±38.2) IEQ vs (586±37.7) IEQ, P＞0.05〕; After purification, there were significant deviation in the islets quantity 〔(408±28.3) IEQ vs (189±27.1) IEQ, P＜0.05〕 and purity quotient 〔（93±2.4）% vs （75±2.1）%, P＜0.05〕. For two groups, there was no obvious deviation of the obtained islets in insulin stimulation and secretion experiment as well as their vivo function experiment. Conclusion The ultimate yield and purity quotient of the rat islets can be obviously improved by using collagenase digestive juice with SIT in situ perfusion on the rat pancreas, and it has no obvious effect on the islets function.
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.
【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.