Experimental Study Of IL-1ra's Prevention On Non-function In Rat Islet Mediated By Inflammatory Cytokines

PrefaceIslet cell separation and purification of the result is a key factor in the success of islet transplantation. The quantity and quality of islet loss depends on a number of complex factors, including the islet separation process in the stress and liver transplantation in parts of the micro-environment. Islet transplantation to the liver, the non-specific cascade of inflammatory material will be activated, including ways to activate the blood clotting and macrophages and endothelial cells to release proinflammatory cytokines (PIC). We assume that completely blocked the IL-lb and its cell receptor binding to isolated pancreatic islets in the PIC trigger apoptosis and necrosis in the islets to provide more complete protection. We have studied culture model of rat islets plus PIC, in order to clarify the mechanism of pancreatic injury and the determination of blocking IL-1ra can provide protection for the injury.Materials and MethodsOrdinary Level closed colony Wistar rats, male and female, weighing 250-280g. Mouse micro-surgical instruments, ultra-clean operating table,0.22um sterilization filter, electric heated water bath (DHW-600), multi-oscillator, electronic balance, micro-plus injector, fluorescence inverted microscope (Olympus), high-speed centrifuge (TDL-5A), CO2 cell incubator (Thermo); fully automated chemiluminescence immunoassay analyzer (Abbott Laboratories AXSYM). Collagenase V-type, Hank's solution, Ficoll-400, dithizone (, DTZ), acridine orange (AO), ethidium bromide (EB); RPMI-1640 culture medium, TNF-), IL-1ra, IL-1β; NO and iNOS detection kit. IFN-γ.T he separation and purification of rat islets.Using the University of Minnesota improved method of separation, purification of rat pancreatic islets. The pancreas in situ perfusion of collagenaseⅤ, fast-cut take, water bath digestion. And then by 80 mesh stainless steel mesh filter, centrifugation. Then using Ficoll discontinuous density gradient centrifugation and purification of isletsIslet culture and groupFreshly isolated islets purified RPMI-1640 complete culture medium 24h. Culture conditions:37℃,5% carbon dioxide,95% air. According to the experimental conditions, were randomly divided into four groups:A group (control group); B group (cytokine); C group (IL-1ra group); D Group:(IL-1ra+cell factor group).Outcom measures Three parameters were observed in vitro glucose-stimulated insulin release test. Acridine orange/ethidium olfactory (AO/EB) fluorescent staining showed islet cell motility. Detection of islet culture medium in the islet tissue iNOS levels of NO.The significant ofdifference was evaluated by q test of ANOVA. P＜0.05 was considered having statisticalsignificance.Results1, pancreatic islet function tests:insulin release test results show, A group of islets after 24h culture, the higher levels of insulin secretion, islet function well. Group B seriously damaged islet function, insulin secretion and insulin release index (SI) decreased significantly (P＜0.01); D group of insulin secretion and SI was significantly higher than those in B group (P＜0.01). C group islets in the culture medium simply by adding AG, the insulin secretion and SI has increased, but no statistically significant (P＞0.05);2, identification of insulin activityA group and C group of islets by the AO/EB staining, visible under inverted microscope, the majority of islet green fluorescence, suggesting that islet survival good. While the B group of the majority of pancreatic islet staining yellow fluorescence, suggesting that islet survival poor, a large number of deaths. Group D islets most of the intact form, issued a green fluorescence, survival significantly better than B group.3, islet culture medium NO levels and iNOS activity in pancreatic islet tissue A group of islets cultured 24h, the culture medium NO concentration and iNOS activity in the organization than those in the lower islet.Group B medium NO concentration in pancreas and islet iNOS activity in the organization as compared with group A was significantly higher (P＜0.01), in particular, iNOS activity of more than A group of more than 10 times; D group of islet iNOS activity in the organization than the B group was significantly inhibited, NO levels are significantly lower (P＜0.01), with the A group of basically the same level, the difference was not statistically significant (P＞0.05); C group of islet iNOS activity and NO concentration in culture medium although lower than the A group and D group, but the difference was not statistically significant (P＞0.05).ConclusionsIn the IL-1β, TNF-α, such as cytokine stimulation, islet tissue inducible nitric oxide synthase (iNOS) expression may be a large number of catalytic synthetic high concentrations of NO is the effect ofβ-cell injury factor. IL-1ra on the islet may be the protective mechanism to prevent IL-1βand other pic with the relevant cell receptor binding and thus play a biological biological effects, blocking cytokine IL-1βand TNF-αon the damage of pancreatic islets, to block its toxic effects on the islet, thus reducing cell damage factor on the islet to improve islet survival and function...