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.
Objective To study the effect of pravastatin on the survival of islet xenografts.MethodsPigtomouse islet transplantation was performed. The models were divided into 4 groups: group A (control); group B, treated with CsA; group C, treated with pravastatin; group D, treatment with combined CsA with pravastatin. The survival time (ST) of the grafts in each group were recorded. Histological examination was used to detect the inflammation and islet cells in the graft. The infiltrated cells were detected by immunohistochemistry with CD4+, CD8+ and CD68 monoclonal antibody. The serum NO was measured. RTPCR was used in the test of IFNγ mRNA.ResultsThe ST of group A,B,C,D was (6.2±0.82) d, (9.2±1.92) d, (7.2±1.30) d, (11.2±1.76) d respectively, the ST of group D was much longer than that of the other groups (P＜0.05).Compared to that in other groups, less infiltrated cell in group D was found. On the 4th postoperative day, the serum NO in group A was (105.0±19.3) mmol/L,significantly higher than that in group B 〔(88.20±21.04) mmol/L〕, in group C 〔(70.7±17.8) mmol/L)〕 and in group D 〔(56.30±16.4) mmol/L〕. When rejection occurred, the serum NO in group C and D was (83.7±10.6) mmol/L and (71.3±13.8) mmol/L, also lower than that in group A (P＜0.05), the serum NO in group B was (104.7±16.3) mmol/L, compared that in group A, no significance was present (Pgt;0.05）. On the 4th postoperative day, the serum expression of IFNγ mRNA in group D was 23.5±4.6, lower than that in group A (28.8±4.8), and no significance was present compared with that in group B and C. ConclusionPravastatin can abate the role of macrophages, especially combined with Cyclosporine, and can prolong the survival of islet xenograft.
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 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 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.
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.
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.
【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.
Objective To explore the effect of ghrelin on insulin secretion and expression of glucose transporter protein-2 （Glut-2） in isolated pancreas of rats. Methods Twenty five Wistar rats were randomly devided into normal control group （NC group）， high concentration of glucose group （HCG group）， high concentration of glucose with high concentration of ghrelin group （10-8mol/L， HCG＋HCGh group）， medium concentration of ghrelin group（10-9mol/L， HCG＋MCGh group）， and low concentration of ghrelin group （10-10mol/L， HCG＋LCGh group） with 5 rats in each group. The rat isolated pancreas perfusion models were established firstly， then from the distal end of abdominal aortas， the models were perfused with low concentration of glucose （5.5mmol/L）， high concentration of glucose （33.3mmol/L） or high concentration of glucose added with different concentrations of ghrelin. Levels of insulin outflowed from portal vein were tested by ELISA method， expression levels of Glut-2 protein were tested by immunohistochemical method，and ultrastructure changes of islet β cell were observed under the transmission electron microscope. Results There were no significant difference on levels of fasting blood glucose （FBG）， fasting insulins （FINS）， homeostasis model of assess-ment for insulin resistence index （HOMA-IR）， and homeostasis model of assessment for pancreatic β cell function （HOMA-β），（P＞0.05）. There were no significant difference on insulin levels of effluent from portal vein of 5 groups （P＞0.05） when isolated pancreas perfused with 5.5mmol/L glucose， while had 2 secretion peaks in 3min and 10-12min after 33.3 mmol/L glucose perfusion， where HCG＋HCGh group at the top. The mean density value of Glut-2 protein in NC group was higher than that of other 4 groups （P＜0.05）. The results of transmission electron microscopy showed that apoptosis was lighter in NC group than that of other 4 groups， and apoptosis of HCG＋HCGh group was lighter than that of HCG＋MCGh group and HCG＋LCGh group. Conclusions In isolated pancreas of rats， ghrelin promotes high concentration of glucose-stimulated insulin secretion， decreases expression of Glut-2 protein， and protects the islet β cell.
Objective To investigate the anti-rejection effect and the mechanism of triptolide （TPT） on islet allo- grafts in a murine model. Methods BALB/c mice were used as islet donor. C57BL/6 mice were rendered diabetic by streptozotocin （STZ） injection， and transplanted with islets under the left kidney capsule. The recipients were randomly （method of random digits table） divided into three groups （n=8）. The mice in the treatment groups were injected intrap-eritoneally with TPT at 50 μg/kg （low-dose TPT group， L-TPT group） or 100 μg/kg （high-dose TPT group， H-TPT group） daily in the first 5 days and then on alternate days until 14 days；while the mice in control group were given vehicles （1% tween 80）. Blood glucose after operation were monitored. The grafts were defined as rejection when two consecutive reading of blood glucose＞20 mmol/L. The left kidney of three recipients in each group were resected for pathological examination. The proportion of CD4＋CD25＋Foxp3＋ regulatory T cells in spleen tissues were tested by flow cytometry. Results The median survival time of islet allografts from the control group， L-TPT group， and H-TPT group were 12.6 days （9-16 days）， 21.4 days （14-27 days） ， and 27.6 days （19-34 days）， respectivly. The percentageof CD4＋CD25＋Foxp3＋regulatory T cells in spleen tissues of three groups were （5.2±0.6）%， （12.0±1.3）%， and（15.7±1.8）%， respectivly. Compared with control group， the median survival time of islet transplantation in mice exte-nded and the proportion of CD4＋CD25＋Foxp3＋ regulatory T cells in spleen tissues increased （P＜0.05）. Conclusions TPT could increase the percentage of CD4＋CD25＋Foxp3＋ regulatory T cells， reduce the rejection after islet transplanta-tion， and prolong the survival time of islet transplantation in mice. The immunosuppressive effect of TPT shows a dose-dependent.