Evaluation of islets derived from human fetal pancreatic progenitor cells in diabetes treatment.

INTRODUCTION: With the shortage of donor organs for islet transplantation, insulin-producing cells have been generated from different types of stem cell. Human fetal pancreatic stem cells have a better self-renewal capacity than adult stem cells and can readily differentiate into pancreatic endocrine cells, making them a potential source for islets in diabetes treatment. In the present study, the functions of pancreatic islets derived from human fetal pancreatic progenitor cells were evaluated in vitro and in vivo. METHODS: Human pancreatic progenitor cells isolated from the fetal pancreas were expanded and differentiated into islet endocrine cells in culture. Markers for endocrine and exocrine functions as well as those for alpha and beta cells were analyzed by immunofluorescent staining and enzyme-linked immunosorbent assay (ELISA). To evaluate the functions of these islets in vivo, the islet-like structures were transplanted into renal capsules of diabetic nude mice. Immunohistochemical staining for human C-peptide and human mitochondrion antigen was applied to confirm the human origin and the survival of grafted islets. RESULTS: Human fetal pancreatic progenitor cells were able to expand in medium containing basic fibroblast growth factor (bFGF) and leukemia inhibitor factor (LIF), and to differentiate into pancreatic endocrine cells with high efficiency upon the actions of glucagon-like peptide-1 and activin-A. The differentiated cells expressed insulin, glucagon, glucose transporter-1 (GLUT1), GLUT2 and voltage-dependent calcium channel (VDCC), and were able to aggregate into islet-like structures containing alpha and beta cells upon suspension. These structures expressed and released a higher level of insulin than adhesion cultured cells, and helped to maintain normoglycemia in diabetic nude mice after transplantation. CONCLUSIONS: Human fetal pancreatic progenitor cells have good capacity for generating insulin producing cells and provide a promising potential source for diabetes treatment.

[The research applications of db/db mouse].

The db/db mice are perfect animal models of type 2 diabetes which have been widely used. The phenotypes of severe obesity, hyperphagia, polydipsia, and polyuria are due to a spontaneous mutation of the leptin receptor (Lepr). The course of the disease is markedly influenced by genetic background, which is more serious in the C57BLKS/J background. And there are many other spontaneous mutations in different sites of Lepr, which produce a series of animal models of obesity, including db(3J)/ db(3J) mice, db(5j)/db(5J) mice, db(pas)/db(pas) mice, Zucker fa/fa rats, and Koletsky fa(k)/fa(k) rats, etc. These rodents appear similar hyperphagia and severe obesity, but different levels of blood glucose, kidney damage and reproductive ability, providing profuse material to investigate the complex function of Lepr. Here we review the history of the discovery of the leptin signaling pathway, the abnormal phenotypes of db/db mice in metabolic, reproductive, immune, etc. Discuss their research applications, reproductive strategy, genotyping guideline, the phenotypic diversity of those animal models with Lepr spontaneous mutation and their mutation patterns, respectively.

[Significance of apolipoprotein A1 as biomarker for early diagnosis and classification of bladder urothelial carcinoma].

OBJECTIVE: To investigate the significance of apolipoprotein (Apo)-A1 in urine as a biomarker for early diagnosis and classification of bladder urothelial carcinoma (BUC). METHODS: Urine samples were divided into four groups: normal control group, benign bladder disease group, low-grade malignant BUC group, and high-grade malignant BUC group. Apo-A1, which showed significantly different expression among the four groups, was selected according to the two-dimensional electrophoresis (2-DE) images of the four groups, and enzyme-linked immunosorbent assay (ELISA) was used to quantify Apo-A1 in the four groups. A receiver operating characteristic (ROC) curve was generated, and the optimal operating points on the ROC curve were found to determine the critical concentrations of Apo-A1 for early diagnosis of BUC and differentiation of low-grade and high-grade malignant BUC. The results were verified clinically, and the specificity and sensitivity were calculated. RESULTS: The 2-DE images showed that that the level of Apo-A1 increased from the normal control grouP to high-grade malignant BUC group. The ELISA showed that there was no significant difference in Apo-A1 level between the normal control grouP and benign bladder disease group, but the Apo-A1 level was significantly higher in the BUC groups than in the normal control grouP and benign bladder disease grouP (P < 0.01); the high-grade BUC grouP had a significantly higher Apo-A1 level than the low-grade BUC grouP (P < 0.01). The BUC patients and those without BUC could be differentiated with an Apo-A1 concentration of 18.22 ng/ml, while the low-grade and high-grade malignant BUC could be differentiated with an Apo-A1 concentration of 29.86 ng/ml. When used as a biomarker, Apo-A1 had a sensitivity of 91.6% (98/107) and a specificity of 85.7% (42/49) for diagnosis of BUC and had a sensitivity of 83.7% (41/49) and a specificity of 89.7% (52/58) for BUC classification. CONCLUSION: Apo-A1 may be a biomarker for early diagnosis and classification of BUC and shows promise for clinical application.

[Preventive effects of Salvia miltiorrhiza on multiple organ edema in the rats of limb ischemia/reperfusion].

OBJECTIVE: To investigate the preventive effects of Salvia miltiorrhiza (SM) on multiple organ edema in the rats which suffered from hind limb ischemia/reperfusion( LI/R). METHODS: Twenty four Wistar rats were randomly divided into 3 groups (n = 8): control group (C group), ischemia/reperfusion group (I/R group ), Salvia miltiorrhiza group (SM group). Referring to Tourniquet method, the model rats which underwent 4 hours ischemia and 4 hours reperfusion of hind limbs were made. Thirty minutes before reperfusion, SM was given to the rats in SM group by tail vein injection at the dose of 5 mL/kg. Accurately weighed one gram of heart, liver, kidney, lung, brain, intestine and skeletal muscle from every animals, weigh these specimens after baking (60 degrees C, 55 hours), calculated the ratio of wet and dry (Wet/Dry,W/D). The levels of interleukin-1 (IL-1) ,interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) in plasma and the contents of Superoxide dismutase (SOD) and malonaldehyde (MDA) were measured. The morphologic changes of skeletal muscle were observed with microscope. RESULTS: It was found that after suffering from ischemia/reperfusion, the W/D of every specimens increased in different degree (P < 0.05, P < 0.01). In plasma, the values of SOD decreased but MDA increased obviously (P < 0.05, P < 0.01). The levels of IL-1, IL-6 ,TNF-alpha-a in plasma increased (P < 0.05, P <0.01). After LI/R, infiltration of inflammatory cells, broaden interstitial around muscle fiber and disordered arrangement of muscle fibers could be seen under microscope. However, Compared with LI/R group, W/D and levels of serum inflammatory factors in SM group were all lower, the values of SOD in plasma increased but MDA in plasma failed down. Pathological changes in skeletal muscle were improved. CONCLUSION: Limb ischemia/reperfusion can lead to multiple organ edema, Salvia miltiorrhiza can prevent the edema in some degree by anti-oxidation and anti-inflammation.

[Effects of taurine on hemorheology of rats with type 2 diabetes].

OBJECTIVE: To observe the effects of taurine on hemorheology and oxidative stress of diabetic rats. METHODS: 40 rats were divided into control group, diabetes group and treatment group at random. Diabetic model was reproduced by intraperitoneal injection of streptozotocin. After having been treated with taurine for 8 weeks, glycosylated hemoglobin (HbA1c) and the serum contents of glucose, superoxide dismutase(SOD), malondialdehyde (MDA) were measured. The changes of hemorheology in different groups were detected respectively. RESULTS: Compared with control group, the content of glucose, MDA and HbA1c in diabetic rats was increased, the activity of SOD was decreased, the levels of whole blood viscosity and the aggregation index of red blood cells and hematocrit were increased and RBC deformability index was decreased in diabetic rats. Moreover, taurine was able to apparently reduce high blood glucose and HbA1c (P < 0.05), markedly elevated the activity of SOD, lowered the content of MDA (P < 0.01); and taurine also could significantly reduce the levels of whole blood viscosity and the aggregation index of red blood cells and hematocrit in the meanwhile, and increase RBC deformability index (P < 0.05 or P < 0.01). CONCLUSION: Taurine could enhance the ability of oxidation resistance, improve blood rheology property in diabetic rats, at the same time it could be beneficial to prevent and cure the development of diabetic blood vessel complication.

[Isolation, purification, and functional evaluation of human pancreatic islet].

OBJECTIVE: To isolate and purify human islet according to the method established by Ricordi and to evaluate the function and safety of these isolated human islets. METHODS: Six pancreases were obtained from human corpses. The islets were isolated by liberase digestion and purificated by Ficoll density gradient centrifugation. The numbers, purity and vitality of the islets were analyzed. The various endocrine cell composition and distribution of the islets were checked by immunofluorescence staining. The glucose-induced insulin secretion was detected by chemiluminescence method. The isolated islets were transplanted under the left renal capsules of 10 streptozocin-induced diabetic nude mice. Twenty days later the left kidneys with transplanted islets of 2 mice with normal blood sugar were resected, and then blood sugar level was observed. An isolated human islet was suspended in RPMI-1066 culture medium for 72 h, then culture of pathogenic micro-organisms, endotoxin and procoagulant activity were detected so as to evaluate the security of the islet products. RESULTS: The mean number of the isolated islets was (229 000 +/- 31 000) islet equivalents (IEQs)/pancreas or (4970 +/- 1620) IEQs/g pancreatic tissue, the mean purity was (59.0 +/- 8.9)%, and the mean vitality was (89 +/- 3)% for the purified islets. Immunofluorescence staining showed that there were 4 types of endocrine cells normally distributed in the islets. The mean insulin stimulation index was 8.1 +/- 4.0 (3.8 - 10.2). The glycemia found in the diabetic nude mice decreased to normal levels from the third day after islet transplantation and maintained normal for over 30 days. The parameters of security in these islet products were under the standard scope. CONCLUSION: Human islets obtained according to Ricordi's method reach the standard for clinical islet transplantation in number, purity, vitality, function, and security.

[The affection and significance of NO on the expression of P-selectin in renal injury following hind limb ischemia/reperfusion in rats].

AIM: To probe into the affection and significance of NO on the expression of P-selectin in renal injury following hind limb ischemia/reperfusion in rats. METHODS: In accordance with the conventional approaches of our department, the model rats were prepared after they were made to undergo 4 hours or ischemia followed by 4 hours of reperfusion of hind limbs. The Wistar rats were divided into four groups randomly: Control group, LI/R group, L-Arg group and L-NAME group. And then in those four groups of Wistar rats, a series of values of measurement were determined such as: Plasma concentrations of nitric oxide (NO), blood urea nitrogen (BUN) and creatinine (Cr). Furthermore, biochemically there came to the assessment of the values including myeloperoxidase (MPO), NO, total nitric oxide synthase (tNOS), inducible NOS (iNOS) and constitutive NOS (cNOS) of renal tissue in different groups. By the methods of electrophoresis and biochemistry, the urine protein was mensurated. The immunohistochemical method was used to detect the expression of P-selectin protein. The morphologic changes were observed with a microscope. RESULTS: After hind limbs had suffered from ischemia/reperfusion for 4 hours, there was the occurrence of a series of results such as in the following which were based on the comparison between plasm of LI/R group and control group. The values of NO, BUN and Cr increased significantly, and the trend of indexes such as NO in renal tissue was similar to that in plasma. The values of MPO, tNOS and iNOS in renal tissue all increased significantly after reperfusion, while cNOS decreased distinctly. The urine protein appeared, especially large molecular weight protein. Renal pathology revealed that after LI/R there were edema and infiltration of polymorphonuclear neutrophil (PMN). Immunohistochemically, the expression of P-selectin was upregulated significantly. Compared with LI/R rats, all injury changes were alleviated in L-Arg group. Morphologic changes were mild. Both the content of urine protein and the percentage of apoptosis cell decreased. The expression of P-selectin was downregulated. In L-NAME group, all injury changes got worse. Immunohistochemical results showed strong positive staining of P-selectin. CONCLUSION: The renal injury after LI/R may relate to the strong expression of P-selectin. NO may have protective affection by decreasing the expression of P-selectin and alleviating the adhesion, aggregation and infiltration of neutrophils.

[Expression NOS in acute lung injury following limb ischemia/reperfusion and its significance in rats].

AIM: To investigate the expression and role of inducible NOS (iNOS) and endothelial NOS (eNOS) in acute lung injury following limb ischemia/reperfusion (4h/4h). METHODS: Wistar rats were randomized into four groups: control group, ischemia/reperfusion (I/R) group, L-Arginine (L-Arg) pretreatment group, Aminoguanidine (AG) pretreatment group. The lung tissue of each group was subjected to assay of content of MDA, MPO, W/D and NO2-/NO3-. The expression of iNOS and eNOS was examined with immunohistological staining. The pulmonary morphologic changes were observed under microscope respectively. RESULTS: The acute lung injury existed after limb ischemia/reperfusion. The eNOS downregulation and iNOS upregulation among I/R, L-Arg and AG groups were observed contrasted to the control group. There was no expressional and statistical difference of iNOS between I/R group and L-Arg group. The expression of eNOS was similar between IR and AG but iNOS expression was downregulated in AG. The parameters of MDA, MPO, W/D and NO2-/NO3- in pulmonary tissue were significantly increased in I/R groups compared with those of the control group. The parameters of L-Arg and AG pretreatment groups in comparison with those of the I/R group showed significantly difference. Based on the results of pulmonary pathology, the congestion and infiltration of inflammatory cells existed obviously in IR group. L-Arg played definite role in militating lung injury and AG might make lung injury aggravated. CONCLUSION: The NO definite production from iNOS is possible to play a competitivly protective role in acute lung injury following limb ischemia/reperfusion and antagonist of iNOS may aggravate the lung injury.

[The protect effect of ischemic preconditioning on the gastric mucosal injury following ischemia/reperfusion of hind limbs of rats].

AIM: To observe the degree of gastric mucosal injury following limb ischemia/reperfusion (LI/R), and to investigate the mechanism of gastric mucosal injury and the protection of ischemic preconditioning (IPC) on gastric mucosal injury. METHODS: The model rats which underwent 4 hours of ischemia and 4 hours of reperfusion of hind limbs were made. Then we respectively observed and determined the histologic lesion score after I/R and IPC + I/R. The gastric barrier mucus in mucus were measured in different groups. The values of MPO, SOD, MDA and XOD in gastric mucosa and the values of MDA, XOD, SOD, LDH in plasma were detected. RESULTS: In the LI/R group, the histologic lesion score increased significantly. The content of gastric barrier mucus in mucus decreased significantly. The value of MPO, MDA, XOD in gastric mucosa and the values of MDA, XOD, LDH in plasma increased remarkably and SOD activity in gastric mucosa and in plasma decreased. However in the IPC group, the histologic lesion score decreased significantly and the content of gastric barrier mucus in mucus increased significantly and the value of MPO MDA XOD LDH in gastric mucosa or in plasma decreased remarkably and the SOD activity increased compared to LI/R group. CONCLUSION: LI/R will lead to the development of stress ulcer, oxygen free radicals play an important role in it. IPC can alleviate the damage of gastric mucosa following ischemia/reperfusion of hind limbs. The decrease of OFR is one of the protection mechanism of IPC.

[A cell model of ischemia/reperfusion injury on skeletal muscle].

AIM: To establish a model of ischemia/reperfusion injury on L-6TG cell. METHODS: Cultured L-6TG cells were divided into 2 groups: control group (C), ischemia/reperfusion group (I/R), LDH in culture fluid, SOD, XOD, free calcium in L-6TG cell and mitochondria respiration were evaluated in each group, the micromorphologic changes were observed with microscope. RESULTS: Compared with control group, after L-6TG cell suffered ischemia 4 hours and reperfusion 4 hours, LDH in culture fluid, XOD, free calcium in L-6TG cell all increased significantly, while SOD in L-6TG cell and mitochondrial respiration decreased, structural damage to L-6TG cell was severe. CONCLUSION: Using mimicking ischemic solution and mimicking reperfusion solution can successfully establish a model of ischemia/reperfusion injury on L-6TG cell.

[The effect of NO, ET-1 on brain injury after hind limbs ischemia/reperfusion in rats].

AIM: To study the roles of nitric oxide (NO) and ET-1 in brain injury after hind limbs ischemia/reperfusion in rats and to investigate the effect of NO/ ET-1 balance on brain injury. METHODS: On a model of the hind limbs ischemia/reperfusion (LI/R) of rats, we used L-Arg(L-arginine, L-Arg), one of the substrates in the process of nitric oxide, aminoguanidine (AG) which inhibits nitric oxide synthase(NOS) and ETA receptor antagonist BQ123, to observe the changes of NO, ET-1, MDA, XOD, SOD, LDH in plasma and tNOS, iNOS, cNOS, NO, ET-1, MDA, XOD, MPO, SOD in brain tissue. RESULTS: Compared with the control group, the content of MDA, XOD, LDH in plasma and MDA, XOD, MPO in brain tissue increased. The activity of SOD decreased (P < 0.01). The content of tNOS, iNOS in brain tissue increased, cNOS decreased (P < 0.01). The content of NO, ET-1 in I/R group in plasma and brain tissue increased, the ratio of NO/ET-1 decreased. The brain injury was deteriorated. After using L-Arg and BQ123, the ratio of NO/ET-1 in plasma and brain tissue increased, the brain injury lightened. Whereas after using AG, the ratio of NO/ET-1 decreased, brain injury became more serious. CONCLUSION: The NO/ET-1 ratio decreased after LI/R, brain injury became more serious.