大鼠胰岛分离条件的优化

Optimizing Conditions for Rat Pancreatic Islets Isolation

目的:优化大鼠胰岛分离纯化的条件,为胰岛移植实验奠定基础。方法:通过胆总管灌注胶原酶P来消化大鼠胰腺,分离胰岛,采用不连续密度梯度Ficoll离心法纯化胰岛,观察胶原酶浓度、消化时间以及大鼠体重对胰岛分离结果的影响。双硫腙染色鉴定胰岛,丫啶橙/碘丙啶染色鉴定胰岛细胞活率,糖刺激胰岛素释放试验评价胰岛功能。结果:胶原酶浓度、消化时间以及大鼠体重对胰岛分离结果有重要影响。1mg/ml胶原酶P在37℃静止消化45分钟条件下,胰岛分离效果最佳,效果较其他酶浓度和消化时间条件下好(P＜0．05)。体重350g的大鼠的胰岛收获量778．33±80．21IEQ/胰腺,而体重250g的大鼠的胰岛收获量655．00±56．56 IEQ/胰腺(P＜0．05)。优化条件下分离的胰岛其纯度＞90%,胰岛细胞活率＞90%,低糖(2．8mmol/L)、高糖(16．7mmol/L)刺激胰岛素释放分别为(5．40±1．75)mIU/L/30IEQ,(12．27±2．55)mIU/L/30IEQ(P＜0．05),刺激指数为2．33±0．29。结论:胶原酶浓度、消化时间以及大鼠体重影响胰岛分离结果,优化分离条件可改善大鼠胰岛分离结果。

Objective： To optimize conditions for rat islets isolation and purification, and lay a foundation for islet transplantation experiment. Methods： Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase P digestion, and purified by discontinuous Ficoll density gradient centrifugation. The protocol was carried out to investigate the influence of collagenase concentration, digestion time, and rat weight on the result of islet isolation. Rat islets were identified by Dithizon staining, and islets viability was assessed by Acridine orange /Propidium iodide staining. Glucose-stimulated insulin secretion was detected using insulin radioimmunoassay kits and used to assess the function of rat islets. Results： The concentration of collagenase, the time of digestion and the weight of the rat were important factors for efficacious isolation of islets. Under the condition of digestion by lmg/ml collagenase P for 45min, the effect of islet isolation was optimal （P〈0.05）. Rat with a higher weight （〉300g） showed a higher islet yield than those with a lower weight （250g） （P〈0.05）. The purity of islets isolated under optimizing conditions was above 900/0, and the viability was above 900/0. The insulin secretion upon 2.8mmol/L and 16.7mmol/L glucose simulation was （5.40±1.75） mIU/L/30IEQand （12.27±2.55） mIU/L/30IEQ respectively （P〈0.05）. The simulation index was 2.33±0.29. Conclusions： The result of islet isolation was concerned with collagenase concentration, digestion time, and rat weight. Optimizing conditions for islets isolation can improve the result of rat islet isolation.