应用两层法保存和运输成人胰腺的实验

Preservation and transportation of adult pancreas with two-layer method

目的:提高胰岛的分离纯化率是胰岛移植成功的关键,其中胰腺的冷缺血时间是主要影响因素之一。探讨并建立供体胰腺长时间(>8h)保存和运送方法。方法:实验于2006-06/2007-02在解放军南京军区福州总医院全军器官移植中心完成,得到医院伦理道德委员会批准。①实验方法:应用两层保存法(University of Wisconsin液/全氟化碳),将25例成人胰腺置于广口瓶中,充氧并盖紧,放入冰筒中送达实验室,按冷缺血时间的长短,分为>8h组(n=17)和≤8h组(n=8)。参照改良后Ricordi方法分离胰岛,将胰腺切片置入消化罐中循环消化。应用连续密度梯度离心法在COBE2991离心机中纯化胰岛组织,并用双硫腙、吖啶橙、碘化丙啶染色。②实验评估:分别进行纯化后胰岛计数、纯度和活率检测,同时检测胰岛对高、低糖的反应性。结果:①冷缺血时间>8h组胰腺的冷缺血时间显著长于≤8h组(P<0.01)。②经双硫腙染色后,两组分离纯化的总胰岛当量、胰岛收获量差异无显著性意义(P>0.05);两组胰岛纯度和活率差异也无显著性意义(P>0.05)。③经低糖、高糖分别刺激后,两组胰岛素释放量及其刺激指数差异均无显著性意义(P>0.05)。结论:应用两层保存法保存和运送供体胰腺,可明显延长保存时间,但对胰岛的分离纯化数量和功能没有明显影响,为胰岛移植远距离保存和运送腺体提供了有利条件。

AIM： High isolation and purification rate are crucial for islet transplantation, and the cold ischemia time of pancreas is one of the influential factors. In this study, the long-time （〉 8 hours） preservation and transportation of adult human pancreas were explored and established. METHODS： The experiment was performed at Organ transplantation Center, Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA from June 2006 to February 2007 with the permission of Hospital Ethics Committee. With two-layer cold storage method （University of Wisconsin solution/perfluorocarbon）, twenty-five pancreata were put into wide mouthed bottle and oxygenated. Total organs were divided into 〉 8 hours group （n =17） and ≤ 6 hours group （n =8） according to their cold ischemia time and isolated with Ricordi method after pancreata were transported to the laboratory. Pancreata were purified with density gradient centrifugation method in COBE 2991 centrifugal machine and stained with dithizone, acridine orange, and propidium iodide. ~lslet equivalent per gram weight of pancreas （IEQ/g）, percent purity and viability of islets were counted. Insulin was detected using ELISA kit after overnight-cultured islets were stimulated with low and high glucose.. RESULTS： （1）The mean of cold ischemia time of group 1 （〉 8 hours） was longer than that of group 2 （≤ 8 hours） significantly （P 〈 0.01）. （2）After dithizone staining, there was no significant difference in total IEQ, IEQ/g, percent purity and viability between two groups （P 〉 0.05）. （3）After stimulated with low and high glucose, the release amount of insulin and stimulated index of two groups were not statistically different （P 〉 0.05）. CONCLUSION： The preservation and transportation using two-layer cold storage method could prolong the time of pancreas, but have no influence on IEQ/g and function of islets. This is conducive to the long distance preservation and transportation of islets for transplantation.