Islet transplantation has the potential to restore normoglycemia and prevent the development of diabetic complications such as retinopathy, nephropathy and neuropathy, and could therefore ve a valuable treatment for diabetic patients. The scarcity of available islets is an obstacle for clinically successful islet transplantation. To resolve the problems, we have examined the two methods, islet transplantation with extracellular matrix1 and in vivo expansion of islets with electrically- transfection of growth factors.
Animals ; Diabetes Mellitus/surgery/*therapy ; Fibronectins/therapeutic use ; Hepatocyte Growth Factor/genetics ; Islets of Langerhans/drug effects/*physiopathology ; *Islets of Langerhans Transplantation ; Male ; Rats ; Rats, Wistar ; *Regeneration ; Transfection

OBJECTIVETo obtain massive human pancreatic islets with modified techniques and evaluation of the islets for the clinical allo-transplantation to treat type I and II diabetes.

METHODS28 consecutive adult human pancreata were isolated with modified automated techniques. Islets were purified using continuous density gradient. The islet yield was counted with international standard known as islet equivalent (IEQ). The function of the isolated islets was evaluated by measuring DNA/insulin ratio, static glucose stimulating test in vitro and transplanting the islets into diabetic nude mice in vivo followed by abdominal glucose tolerance test and C peptide measurement.

RESULTSThe yield of 28 consecutive human pancreata isolations ranged from 5 000 to 1 030 000 IEQs/pancreas with the average of 291 635 IEQs/pancreas. The first 13 isolations yielded 49 123 IEQs/pancreas, 846 IEQs/g and, purity 87% in average. The remained 15 isolations after the modifications yielded 501 813 IEQs/pancreas, 7 003 IEQs/g and purity 89% in average. The results of in vitro SGS showed good response to the different glucose concentration. 34 diabetic nude mice were transplanted under the renal capsule with the freshly isolated islets. 29 out of 34 diabetic mice obtained normoglycemia within 12 hours and the glucose tolerance tests were near normal. Serum C peptide level of transplanted mice is close to that of the control group.

CONCLUSIONSMassive human islets can be isolated with the modified techniques. Quality assessment of these islets both in vitro and in vivo has indicated that these high quality human islets could be used for the clinical allogeneic islet transplantation.

Adult ; Animals ; Cell Separation ; methods ; Diabetes Mellitus, Experimental ; surgery ; Glucose ; Humans ; In Vitro Techniques ; Islets of Langerhans ; cytology ; drug effects ; physiology ; Islets of Langerhans Transplantation ; Mice ; Mice, Nude ; Transplantation, Heterologous

OBJECTIVETo explore the effect of Jiangtang Xiaozhi capsule (JXC) on morphological changes of islets and liver at rat model of type 2 diabetic mellitus and provide the experimental basis for the clinical therapy of type 2 diabetic mellitus.

METHODWister rats were fed on a diet enriched in fat and glucose to induce insulin resistan, the rats were injected intrapertoneally with a low-dose streptozotocin (STZ) twice (25 mg x kg(-1)) to induce hyperglycemia, so the successful rat model of type 2 diabetes were established. The experimental rats were divided into model group, high dose JXC group, middle dose JXC group, low dose JXC group, Erjiashuanggua group, Jinqijiangtang group and normal control group. After all the treatment groups received their own medicine for two months, all the rats were sacrificed and morphological examination on their islets and livers were performed.

RESULTFatty liver in various degrees was seen in the model group and all the treatment groups, but the liver steatosis in middle and low dose JXC groups was significantly milder than that in model group (P < 0.05). Islets in the high dose JXC group were significantly more than that in the model group (P < 0.05).

CONCLUSIONJXC can improve significantly the pathological change in islets and liver steatosis at rat model of type 2 diabetic mellitus.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Female ; Islets of Langerhans ; drug effects ; pathology ; Liver ; drug effects ; pathology ; Male ; Microscopy ; Rats ; Rats, Wistar

BACKGROUNDImproving islet graft revascularization has become a crucial task for prolonging islet graft survival. Endothelial cells (ECs) are the basis of new microvessels in an isolated islet, and EC coating has been demonstrated to improve the vascularization and survival of an islet. However, the traditional method of EC coating of islets has low efficiency in vitro. This study was conducted to evaluate the effect of a polyglycolic acid (PGA) scaffold on the efficiency of islet coating by ECs and the angiogenesis in the coated islet graft.

METHODSA PGA fibrous scaffold was used for EC coating of islet culture and was evaluated for its efficiency of EC coating on islets and islet graft angiogenesis.

RESULTSIn in vitro experiments, we found that apoptosis index of ECs-coating islet in PGA group (27% ± 8%) was significantly lower than that in control group (83% ± 20%, P < 0.05) after 7 days culture. Stimulation index was significantly greater in the PGA group than in the control group at day 7 after ECs-coating (2.07 ± 0.31 vs. 1.80 ± 0.23, P < 0.05). vascular endothelial growth factor (VEGF) level in the PGA group was significantly higher than the coating in the control group after 7 days culture (52.10 ± 13.50 ng/ml vs. 16.30 ± 8.10 ng/ml, P < 0.05). Because of a tight, circumvallated, adhesive and three-dimensional growth microenvironment, islet cultured in a PGA scaffold had higher coating efficiency showing stronger staining intensity of enzyme than those in the control group after 14 days of culture following ECs-coating. For in vivo study, PGA scaffold significantly prolonged the average survival time of EC-coated islet graft after transplantation compared with control group (15.30 ± 5.60 days vs. 8.30 ± 2.45 days, P < 0.05). The angiogenesis and area of survived grafts were more in the PGA group compared with the control group by measuring the mean microvessel density (8.60 ± 1.21/mm2 vs. 5.20 ± 0.87/mm2, P < 0.05). In addition, expression of VEGF and tyrosin-protein kinase receptor (Tie-2) gene increased in PGA scaffold group than that in control group by real-time reverse transcription-polymerase chain reaction analysis.

CONCLUSIONSThese results demonstrate that the efficiency of EC coating of islets was successfully increased by culturing ECs on a PGA scaffold. This method enhances the function, survival, and vascularization of isolated islets in vitro and in vivo.

Animals ; Apoptosis ; drug effects ; Endothelial Cells ; drug effects ; Enzyme-Linked Immunosorbent Assay ; Graft Survival ; drug effects ; Insulin ; metabolism ; Islets of Langerhans ; drug effects ; Islets of Langerhans Transplantation ; methods ; Neovascularization, Physiologic ; drug effects ; Polyglycolic Acid ; chemistry ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Tissue Scaffolds ; chemistry

OBJECTIVETo study the effects of a self-formulated blood glucose-adjusting recipe on blood glucose and pathology of the pancreas in diabetic mice.

METHODSDiabetes mellitus (DM) was induced in mice by injection of alloxan through the caudal vein. The blood glucose-adjusting recipe was administered in the mice at the doses of 20, 10 and 5 g/kg for 21 days, after which blood glucose was determined and the sugar tolerance was evaluated, and the pancreas and islet pathologies were examined microscopically.

RESULTSThe blood glucose-adjusting recipe at 20 g/kg significantly lowered the fast blood glucose in the mice, and at 20 and 10 g/kg, the recipe significantly lowered the blood glucose 2 h after glucose administration and increased the sugar tolerance. The pathological damage in the diabetic mice was alleviated after treatment with the recipe.

CONCLUSIONSThe blood glucose-adjusting recipe has a good effect in stabilizing blood glucose in mice.

Animals ; Blood Glucose ; drug effects ; Diabetes Mellitus, Experimental ; blood ; drug therapy ; pathology ; Drugs, Chinese Herbal ; therapeutic use ; Islets of Langerhans ; pathology ; Mice ; Phytotherapy

Nonspecific inflammatory response is the major cause for failure of islet grafts at the early phase of intraportal islet transplantation (IPIT). Bilirubin, a natural product of heme catabolism, has displayed anti-oxidative and anti-inflammatory activities. The present study has demonstrated that bilirubin protected islet grafts by inhibiting nonspecific inflammatory response in a syngeneic rat model of IPIT. The inflammation-induced cell injury was mimicked by exposing cultured rat insulinoma INS-1 cells to cytokines (IL-1beta, TNF-alpha and IFN-gamma) in in vitro assays. At appropriate lower concentrations, bilirubin significantly attenuated the reduced cell viability and enhanced cell apoptosis induced by cytokines, and protected the insulin secretory function of INS-1 cells. Diabetic inbred male Lewis rats induced by streptozotocin underwent IPIT at different islet equivalents (IEQs) (optimal dose of 1000, and suboptimal doses of 750 or 500), and bilirubin was administered to the recipients every 12 h, starting from one day before transplantation until 5 days after transplantation. Administration of bilirubin improved glucose control and enhanced glucose tolerance in diabetic recipients, and reduced the serum levels of inflammatory mediators including IL-1beta, TNF-alpha, soluble intercellular adhesion molecule 1, monocyte chemoattractant protein-1 and NO, and inhibited the infiltration of Kupffer cells into the islet grafts, and restored insulin-producing ability of transplanted islets.
Animals ; Apoptosis/drug effects/immunology ; Bilirubin/*administration & dosage/pharmacology ; Cell Line, Tumor ; Cytokines/immunology/metabolism ; Diabetes Mellitus, Experimental/*drug therapy/*immunology ; Inflammation ; Inflammation Mediators/immunology/metabolism ; Islets of Langerhans/drug effects/*immunology/injuries/pathology ; *Islets of Langerhans Transplantation ; Male ; Oxidative Stress/drug effects/immunology ; Rats ; Rats, Inbred Lew ; Transplantation, I

BACKGROUNDIslet transplantation represents an ideal therapeutic approach for treatment of type 1 diabetes but islet function and regeneration may be influenced by necrosis or apoptosis induced by oxidative stress and other insults. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide. It has also been reported to be an antioxidant enzyme which can improve the function of grafted islets by cytoprotection via free radical scavenging and apoptosis prevention. In the present study, we investigated whether transduction of HO-1 genes into human islets with an adenovirus vector has cytoprotective action on islets cultured in vitro and discuss this method of gene therapy for clinical islet transplantation.

METHODSCadaveric pancreatic islets were isolated and purified in vitro. Transduction efficiency of islets was determined by infecting islets with adenovirus vector containing the enhanced green fluorescent protein gene (Ad-EGFP) at multiplicities of infection (MOI) of 2, 5, 10, or 20. Newly isolated islets were divided into three groups: EGFP group, islets transduced with Ad-EGFP using MOI = 20; HO-1 group, transduced with adenovirus vectors containing the human HO-1 gene using MOI = 20; and control group, mock transduced islets. Insulin release after glucose stimulation of the cell lines was determined by a radioimmunoassay kit and the stimulation index was calculated. Flow cytometry was used to detect apoptotic cells in the HO-1 group and in the control group after induction by recombinant human tumor necrosis factor-alpha (rTNFalpha) and cycloheximide (CHX) for 48 hours.

RESULTSAdenovirus vectors have a high efficiency of gene transduction into adult islet cells. Transduction of islets with the Ad-EGFP was most successful at MOI 20, at which MOI fluorescence was very intense on day 7 after transduction and EGFP was expressed in cultured islet cells for more than four weeks in vitro. The insulin release in the control group was (182.36 +/- 58.96) mIU/L after stimulation by high glucose media (16.7 mmol/L), while insulin release from the HO-1 group and the EGFP group were (270.09 +/- 89.37) mIU/L and (175.95 +/- 75.05) mIU/L respectively. Compared to the control group and the EGFP group, insulin release in the HO-1 group increased significantly (P < 0.05). After treatment with rTNFalpha and CHX the apoptotic ratio of islet cells was (63.09 +/- 10.86)% in the HO-1 group, significantly lower than (90.86 +/- 11.25)% in the control group (P < 0.05).

CONCLUSIONSTransduction of human islets with Ad-HO-1 can protect against TNF-alpha and CHX mediated cytotoxicity. The HO-1 gene also appears to facilitate insulin release from human islets. Transduction of donor islets with the adenovirus vector containing an HO-1 gene might have potential value in clinical islet transplantation.

Adenoviridae ; genetics ; Apoptosis ; drug effects ; Cycloheximide ; pharmacology ; Cytoprotection ; Genetic Therapy ; Heme Oxygenase-1 ; genetics ; physiology ; Humans ; Insulin ; secretion ; Islets of Langerhans ; physiology ; Transduction, Genetic ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo establish an semi-automated effective method for large-scale purification of islet cells from human pancreas.

METHODSHuman pancreas tissue was digested with collagenase P using a semi-automated pancreas-digestion system followed by purification in a HCA-Ficoll continuous gradient using Cobe2991 cell separator. After isolation, the islet cell yield and purity was evaluated with light microscope with DTZ staining, and the islet function assessed by insulin release assay in vitro.

RESULTSThe number of the islets collected from each pancreas averaged 38 6201-/+78 219 islet equivalents (IEQ) before purification, and 231 420-/+28 054 IEQ after the purification with discontinuous gradient centrifugation. From each gram of the pancreatic tissue, 3148-/+317 IEQ were obtained with an average purity of (62.81-/+2.68) %. The purified islets responded well to high-concentration (16.7 mmol/L) glucose stimulation with a 2.53-fold increase of insulin secretion over the basal level (3.3 mmol/l, P<0.001).

CONCLUSIONThe established semi-automated method can be applicable for large-scale purification of fully functional islet cells from human pancreas.

Cell Count ; Cell Separation ; instrumentation ; methods ; Cell Survival ; Glucose ; pharmacology ; Humans ; Insulin ; secretion ; Islets of Langerhans ; cytology ; drug effects ; metabolism ; Reproducibility of Results

AIMTo isolate, culture pancreatic progenitor cells derived from islets of newborn rats, and to observe the effect of GLP-1 (7-36) NH2 on islet progenitor differentiation.

METHODSIslets were isolated purified, islet progenitor cells were isolated and proliferated in the modified RPMI-1640 medium supplemented with 20 microg/L bFGF and 20 microg/L EGF, then were differentiated with 20 nmol/L GLP-1 (7-36) NH2. The properties of islet progenitor cells were identified primarily by hybridization in situ, immunocytochemistry, dithizone (DTZ)-staining, and radioimmunoassay before and after differentiation.

RESULTSIslet progenitor cells did not express PDX-1, insulin and somatostatin, but nestin. After differentiation, a portion of cells expressing PDX-1, insulin mRNA, insulin, somatostatin, and nestin, islet-like cell clusters (ICCs) were formed, DTZ-stained cells were in peripheral region of it. Insulin release was markedly greater in media harvested after differentiation of 3 weeks.

CONCLUSIONA kind of progenitor cells exists in pancreatic islet of newborn SD rats, could be expanded continuously. GLP-1 (7-36) NH2 could differentiate pancreatic islet-derived progenitor cells to form ICCs capable of insulin secretion.

Animals ; Animals, Newborn ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Separation ; Cells, Cultured ; Glucagon-Like Peptide 1 ; pharmacology ; Islets of Langerhans ; cytology ; Male ; Rats ; Stem Cells ; cytology

OBJECTIVETo evaluate the insulin and leptin resistance of curcumin on simplicity obesity rats.

METHODSAll 50 SPF grade healthy Sprague-Dawley male initial weaning rats were used for two groups in stratified sampling by weight: 30 in treated group and 20 in control group. They were assigned to the following treatment for 8 weeks: the treated group was fed with high-fat food and the control group was fed with normal food. Eight weeks later, adiposity model rats were prepared. Groups: adiposity model rats were divided into 3 groups: model + low curcumin (1.25 g/kg), model + high curcumin (5.00 g/kg) and a model group. In addition, there also had a normal control and a control + high curcumin (5.00 g/kg) group. Ten rats in every group and all given ground feed. After intragastric administration in different doses of curcumin 4 weeks, the effects and pathological changes were observed by the blood sugar, insulin, leptin and TNF-alpha, pathology and transmission electron microscope of pancreatic gland.

RESULTSGiven 4 weeks the different dose of curcumin on the simplicity obesity rats, the significant diminished weight (435.0 +/- 37.6) g and content of lipocyte (4.78 +/- 1.87) g as compared with the obesity model control (492.3 +/- 14.8) g and (8.94 +/- 1.88) g (t values were 4.484 and 4.961 respectively, P < 0.01), level of blood sugar (4.50 +/- 0.09) mmol/L, insulin (7.43 +/- 0.65) mmol/L, leptin (3.40 +/- 0.39) mmol/L and TNF-alpha (2.42 +/- 0.19) ng/ml were significantly decreased than those of adiposity model rats (4.94 +/- 0.12) mmol/L, (9.30 +/- 0.21) mmol/L, (4.40 +/- 0.23) mmol/L and (2.86 +/- 0.49) ng/ml (t values were 8.297, 7.743, 6.247 and 2.368 respectively, P < 0.05), and there was no significant difference with the control group (4.30 +/- 0.14) mmol/L on the level of blood sugar (t = 0.399, P > 0.05). There were a lot of secretory granules with large sphere volume in beta cells of pancreatic island found by transmission electron microscope, and these secretory granules had a higher electron density than those in non-disposed groups.

CONCLUSIONBy diminishing the sediment of fat, relaxing the lymphatic return, and refraining the apoptosis of beta cells, the curcumin might significantly decrease the level of insulin resistance and leptin resistance caused by the high fat diet.

Animals ; Apoptosis ; Curcumin ; pharmacology ; Disease Models, Animal ; Insulin ; metabolism ; pharmacology ; Insulin Resistance ; Islets of Langerhans ; drug effects ; Leptin ; metabolism ; pharmacology ; Male ; Obesity ; metabolism ; Rats ; Rats, Sprague-Dawley