冷缺血时间对大鼠胰岛结构和功能的影响

目的 探讨胰腺的冷保存时间对胰岛的获得量、活性、纯度及功能的影响.方法 采用Wistar大鼠,切取其胰腺,然后保存于4℃UW液中,分别于保存3 h(3 h组)、6 h(6 h组)、9h(9 h组)、12 h(12 h组)、15 h(15 h组)、18 h(18 h组)和21 h(21 h组)取出胰腺,采用明尼苏达大学改良方法分离、纯化胰岛,并设不经过冷保存的对照组.分离、纯化所得的胰岛以双硫腙染色,分析胰岛数量与纯度;丫啶橙/溴化乙啶染色分析胰岛活性;测定胰岛在葡萄糖刺激下的胰岛素分泌量.结果 对照组每个大鼠胰腺平均叮获得560当量胰岛,形态完整.纯度达到88%,活性达到94%.3 h组和6 h组的胰岛获得量略有下降,但与对照组相比,差异无统计学意义.9 h组、12 h组、15 h组、18 h组和21 h组的胰岛获得量较对照组、3 h组和6 h组明显下降(P＜0.01,P＜0.05),各组间相比较,差异也有统计学意义(P＜0.01),且随保存时间的延长,各组的胰岛活性和纯度也逐渐下降.9 h组、12 h组和15 h组胰岛细胞形态基本完整,但可见少数细胞被膜不完整,内分泌颗粒外溢.18 h组和21 h组胰岛形态不规则,被膜破裂,大量内分泌颗粒外溢.对照组、3 h组和6 h组对高糖刺激反应良好,三者问的胰岛素释放指数(SI)的差异无统计学意义,保存9 h以后,各组SI均较对照组、3 h组和6 h组明显下降(P＜0.01,P＜0.05),且各组间的差异也有统计学意义(P＜0.01),18 h组和21 h组尤其明显.结论 采用UW液低温保存大鼠胰腺6 h以内,获得的胰岛的数量较多,质量较好;保存6～15 h,获得的胰岛数量及质量有所下降,但仍然可以用于移植;保存时间超过15 h,获得的胰岛数量及质量明显下降,不宜用于移植.

Influence of cold ischemic time on rat islet structure and function

Objective To investigate the effect of cold ischemic time on quality and yield of islets isolated from rat pancreas.Methods Wistar rats were divided into 8 group aecording to the cold ischemic time before isolation:0 h group(control group),3 h group,6 h group,9 h group,12 h group,15 h group,18 h group,21 h group.Islets were isolated and purified according to the modified Minnesota program.The quantity and the degree of purity were measured by dithizone staining.The viability was tested by AO/EB staining.Insulin secretion in response to glucose challenge in vitro was determined.Results 560 IE islets were procured from one donor rat averagely in the control group,and the degree of purity was 88%and the viability was 94%.The islet quantity,viability and in vitro function showed no differenee between 3 h or 6 h groups and control group.The quantity and viability were decreased after preservation for more than 9 h.The yield of the islets in 9 h group,12 h group,15 h group and 18 h group were decreased as compared with the control group(P＜0.01,P＜0.05),and there was significant difference among the groups.The yield and function of islets was decreased with prolongation of cold ischemic time.In the 9 h group,1 2 h group and 1 5 h group,the structure of most islets were basically normal,only few islets showed ruptured membrane with endocrine granules leakage.In 18 h group,the purity was 43%and the viability was 36%,which did not meet the clinical criteria.The in vitro function showed the sarne trend.The SI of 18 h and 21 h groups was less than 1.5.The structure of the most islets in these two groups was abnormal.Conclusion The islet vield and function remained perfect after 6 h preservation.Longer cold ischemic time reduced the yield and function,and after preservation for 15 h,the islet graft was not available for transplantation.