大鼠胰岛分离和纯化

Isolation and purification of rat islets

目的观察胶原酶消化法对大鼠胰岛的分离结果.方法用胶原酶法分离、Ficoll400 密度梯度离心法纯化胰岛.用DTZ法判定胰岛的纯度;用AO/PI法判定胰岛存活率;用胰岛素释放试验和移植糖尿病大鼠法判定胰岛功能.结果纯化后的胰岛收获量为(500-700)个/每只胰腺,胰岛纯度大于85%,活度大于95%,胰岛细胞体外培养液中胰岛素含量在基础相和刺激相为(18.32±3.57)mu/L、(32.25±3.74)mu/L,差异有显著性(P＜0.05),胰岛移植后,实验组2天后血糖浓度降至正常,对照组无改变,实验组正常血糖维持时间(5.1±2.4)d.结论胶原酶消化、Ficoll400密度梯度离心法可获得高纯度和活力的大鼠胰岛.

Objective:To observe the digestion effects of collagenase on rat pancreas.Methods:Islets were isolated using digestion by ductal injection of collagenase,and purified by centrifugation of Ficoll density gradient.The identification of purified islets was evaluated by dithizone staining.The viability of islet was as-sessed by fluorescence staining of aridine orange(AO)and propidium iodide(PI).The function of purified islets was determined by Glucose-stimulated insulin release test and transplantation of rat with streptozocin-induced diabetes.Results:The total number of purified islets of one pancreas was from500to700.The pu-rity of islets was more than85percent.The viability of islets was more than95percent.The secretion amount of insuline was(18.32±3.57)mu/L and(32.35±3.74)mu/L at fundamental and stimulated phase,re-spectively.There was significant difference between the two phases(P<0.05).The concentration of blood sugar recovered to normal level after two days in the group of islet transplantation.But there was not any changes in the concentration of blood sugar in the control group.The mean time in the experimental group was(5.1±2.4)days.Conclusion:The digestion of collagenase for rat has a high yield and high purity of islets which have biological vigor.