| PrefaceGood prospects for the development of islet transplantation,it is still faced with the need for pancreatic mass,a lower survival rate,recurrence of autoimmune diseases caused by graft failure and so on.Many factors affect the effectiveness of islet transplantation,the pancreas has been the ischemic time be considered one of the most important factor.Rat and human islets in many ways share the same characteristics as the number of cases of pancreas transplantation increases every year,non-heart-beating donor(non-heart-beating donor,NHBD)the use of organs has become a hot issue, NHBD inevitably experience for pancreatic a period of warm ischemia injury,to explore for the safety of pancreatic warm ischemia time limit injury and graft function and postoperative status of the relationship has great significance.However,so far for the tolerance of pancreatic warm ischemia time limit is still a lack of conclusive,the Japanese scholars believe that the safety of warm ischemia time limit＜30 minutes; American scholars think＜15 minutes.This experiment is to investigate the effects of different warm ischemia time of rat islet isolation and purification results of the clinical islet transplantation for those who provide the basis for selection.Materials and MethodsExperimental animals and grouping:This experiment used male Wistar rats (Laboratory Animal Center,Chinese Medical University to provide),weighing 250~ 300g.Weighing rats after intraperitoneal injection of anesthesia.Median abdominal incision into the abdomen.Cut diaphragm with vascular clamp at the bottom of the base closure resulting in cardiac arrest the heart,blocking the thoracic aorta,resulting in non-heart-beating donor(NHBD)model of warm ischemia.Warm ischemia time in accordance with the different rats were randomly divided into 4 groups of 10.In rat-heart-beating,the first group 1 to group 4,respectively,0 minutes,15 minutes,30 minutes,45 minutes to 4℃UW solution preservation of 8h,and then 10 ml collagenase solution injected into the pancreas.Harvesting islet:Flush the rat pancreas by UW solution in situ vascular,cut itdown,preserve with UW solution at 40C.Retrograde perfusion of collagenase solutioninto the rat pancreas duct,fully inflate the pancreas.Digestion by vibration in the water bath of 3711 0℃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 of25%,23%,20.5%and 11%.Aspirate the 11%and 20.5%Ficoll solution containingislet.Observed index:DTZ stain is used to decide the number and purity of the isolatedislet.AO/EB fluro-stain showed the viability of cell and the insulin secretion trial bythe ex vivo glucose stimulation showed the islet viability.Immunohistochemical stainof Rabbit Anti-Insulin were used to determine the expression of insulin of islet.Statistical analysis:all the data expressed in mean1SD.The significant ofdifference was evaluated by q test of ANOVA.P＜0.05 was considered having statisticalsignificance.ResultsNHBD rat warm ischemia time at 30 minutes in each group to obtain the number of islet purity,viability and activity when compared with the 0 min group was no significant difference(P＞0.05).NHBD rat warm ischemia time was at 45-minute group the number of islet purity,viability and activity when compared with 0 minutes there is significant difference between groups(P＜0.05),lower than that of islet transplantation on clinical criteria(activity＞75%,purity＞50%).Conclusions1.warm ischemia time of 15 minutes of NHBD rats had no effect on islet isolation and purification.2.warm ischemia time at 15-30 minutes,the NHBD rat islets of no significant impact on separation and purification,can be used for islet transplantation.3.warm ischemia time more than 30 minutes,the NHBD rat islet isolation and purification can have significant impact,should not be used for islet transplantation.
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