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 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 identify pancreatic duct-derived stem cells （PDSCs） from the pancreatic duct of rats, and culture in three-dimension cell culture system or two-dimension cell culture, with characterization on their morphology and phenotype. Methods Adult male Wistar rats underwent perfusion with collagenaseⅤvia the pancreatic duct, the pancreas was surgically procured, digested, followed by discontinued density gradient centrifuge to isolate islets from acinar and ductal tissue. The acinar and ductal tissues were cultivated in serum-containing medium in the three-dimension or two-dimension cell culture seperatedly to obtain adherent cells, as PDSCs, which were expanded by consecutive passages. The cell junction, cell cycle, and cell apoptosis rate between the two-dimension and three-dimision cell culture were compared by means of microscope and flow cytometry. Results PDSCs of rats showed direct adherence culture in three-dimension and two-dimision cell culture systerm. PDSCs grew in many layers in three-dimension cell culture system on 2 days after culture, and possessed capacity of high proliferation on 7 days. Cells were adherent to the bottom of flask on 5 days after culture in two-dimision cell culture. Cell junctions were more obvious in three-dimension cell culture system. Compared with the two-dimision cell culture, the cells of G1 phase increased〔（56.46±1.17） % vs.（37.33±1.21）%〕, and the cells of G2 phase decreased〔（14.62±0.15） % vs. （32.40±1.21）%〕in three-dimension cell culture system （P＜0.01）. The early period apoptosis rate was lower in three-dimension cells culture system than that in two-dimision cell culture 〔（1.48±0.39）% vs. （5.24±1.33）%, P＜0.05〕. The late period apoptosis rate was no statistically significant difference between the three-dimension and two-dimension cell culture （P＞0.05）. Conclusions PDSCs of rats could be obtained in the three-dimension cell culture system. There is cell junction in three-dimension culture mode, which shows a significant enhancement on intercellular interaction, and the biological characters are different between the three-dimension and two-dimension cell culture.
【Abstract】Objective To investigate the effects of preservative temperature on pancreatic function and determine the optimal preservative temperature for pancreatic transplantation. MethodsCold pancreatic preservation was performed and a homologous male Wistar rat model of heterotopic total pancreaticoduodenal transplantation was established. The pancreas was preserved for 6 h in UW solution at specific temperatures(0 ℃, 4 ℃, 8 ℃ and 12 ℃). After preservation, pancreatic tissue was taken for histologic examination in every group. ATP and total adenine nucleotides (TAN) were determined by using high performance liquid chromatography (HPLC). Blood glucose(BG), serum amylase and lipase were measured 24 h after transplantation. And the activity of myeloperoxidase (MPO) in the pancreatic grafts was also measured at the same time. Besides, histological observation was performed. Results Microscopic studies showed that the histomorphological changes of pancreas in 4 ℃ group were less obvious than those in other groups. Tissual concentrations of ATP and TAN decreased gradually in 4 ℃ group, 0 ℃ group, 8 ℃ group, and 12 ℃ group after 6 h of preservation(PH＜0.05). The levels of BG, serum lipase and MPO increased gradually in 4 ℃ group, 0 ℃ group, 8 ℃ group, and 12 ℃ group(PH＜0.05). The activity of MPO in 4 ℃ group （1.19±0.16 U/g ）was significantly lower than that of the control group（0.26±0.09 U/g，PH＜0.05). Conclusion The temperature of 4 ℃ is most appropriate for hypothermic pancreatic preservation and can considerably alleviate cold ischemic injury of rat pancreas.