Effect Of Cold Ischemia Time To The Isolation And Purification Of Rat Islet

Effect of cold ischemia time to the isolation and purification of Rat isletPrefaceAs one of the common endocrine diseases, DM can lead to kinds of complications threatening health of patients. Building an endogenous insulin-secretion system is able to curc the DM permanently. Nowadays only pancreas or islets transplant fulfills the purpose. The islet transplant has the advantages including: safer, simpler, more convenient for the modifying in vitro, lower rate of side-effect and easier to rcpcat than the pancreas transplant. The islet transplant has become a prospective method. Adequate and high-quality islet is required by successful islet transplantation. The time of pancreas by cold storage has a strong connection with the ability to recover viable islet. UW solution is one most widespread and effective pancreas preserved solution now. Several clinical studies have demonstrated that pancreas can hypothcrmically preserved 8-9 hours for islet transplantation. Our trial preserved pancreas of rat with UW solution at 4℃to different hours before islet isolation and investigated the effect of cold ischemia to the number and viability of islet.Materials and MethodsAnimal: SD rat of either sex with weigh ranging from 250-300g as the donor, provided by animal center of China medical university, which are randomly divided into 8 groups for preserved different hours, every group contained 5 rats.Harvesting islet: Flush the rat pancreas by UW solution in situ vascular, cut it down, preserve with UW solution at 4℃. Retrograde perfusion of collagenase solution into the rat pancreas duct, fully inflate the pancreas. Digestion by vibration in the water bath of 37±1℃for 12-15 min. At the end of digestion, double centrifuge and filter by 80 aperture grit. Purifying islet by Ficoll graded centrifuge at concentration of 25%, 23%, 20.5% and 11%. Aspirate the 11% and 20.5% Ficoll solution containing islet.Observed index: DTZ stain is used to decide the number and purity of the isolated islet. AO/EB fluro-stain showed the viability of cell and the insulin secretion trial by the ex vivo glucose stimulation showed the islet viability. Immunohistochemical stain of Rabbit Anti-Insulin were used to determine the expression of insulin of islet.Statistical analysis: all the data expressed in mean±SD. The significant of difference was evaluated by q test of ANOVA. P＜0.05 was considered having statistical significance.ResultsWhen cold preservation was less than 6 hours, all the groups had no significant diversity in amount, purity, survival rate and activity of islets isolated from pancreas whether:they were preserved in UW solution or not (P＞0.05).But when the cold preservation time was beyond 6 hours, there were significant difference between the preserved groups and the control one (P＜0.05). Furthermore, it showed obvious time-dependent relation, that racreasing the duration of cold storage prior to islet isolation decreased measures of islet amount and viability. When cold storage was more thanl5 hours, all the amount, purity, survival rate and activity of islets were significantly reduced, cold preservation for 18 hour, islet isolation were successful in amount 171 IEQ at purity 44% and survival rat 41%.Conclusions1,When cold preservation time in UW solution is less than 6 hour, the quantity and quality of islets isolated from pancreas of rat is beneficial.2,The duration of preservation is from 6 to 15 hours, more time, less amount and worse quality of islet, but it can still be used.3,When the rat pancreas is preserved for more than 15 hour, it is unsuitable for islet transplantation.