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.
ObjectiveTo study the characteristics of the human umbilical cord perivascular cells (HUCPVC) isolated from human first trimester umbilical cord perivascular layer tissues and the differentiation into islet-like cell clusters in vitro. MethodsThe HUCPVC derived from human first trimester umbilical cord which was donated by the volunteers were isolated and subcultured. The surface markers such as stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, OCT-4, TRA-1-60, and TRA-1-81 were detected by immunohistochemical method. The first trimester HUCPVC were induced to embryoid bodies (EB)-like cell aggregations and islet-like cell clusters in vitro through a simple stepwise culture protocol (5 steps). The expressions of specific markers[α-fetoprotein (AFP), Nestin, and smooth muscle actin (SMA)] were measured by immunohistochemical method; and the ability of glucose-stimulated insulin secretion was analyzed. ResultsThe first trimester HUCPVC were successfully isolated and could be passaged steadily more than 10 generations, which expressed SSEA-3, SSEA-4, OCT-4, TRA-1-61, and TRA-1-81. The first trimester HUCPVC were successfully induced into EB-like cell aggregations and islet-like cell clusters. The EB-like cell aggregations could express markers of three germ lineages:AFP, Nestin, and SMA. The islet-like cell clusters could release insulin significantly in response to elevated concentrations of glucose in vitro (t=7.444, P=0.002). The insulin contents were (23.2±5.3) mU/L and (7.0±0.5) mU/L in high and low glucose media, respectively. ConclusionThe first trimester HUCPVC has the ability to differentiate into islet-like cell clusters which can secret insulin 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.
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 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 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.
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 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.
【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 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.