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.
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.
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.
【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 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.
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 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.
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 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 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.
