大鼠胰岛分离纯化及功能测定

目的探讨分离和纯化高纯度、高活率、功能佳的大鼠胰岛的方法，并为胰岛移植等实验建立基础。方法采用胶原酶P逆行胆总管灌注，消化分离成年大鼠胰岛，Ficoll 400非连续密度梯度离心法纯化胰岛。形态观察，双硫腙（DTZ）染色鉴定胰岛，AO／PI染色检测细胞活率，胰岛素释放试验评价胰岛的功能。结果分离纯化后大鼠胰岛形态完整，平均获得量为（512±89）IEQ，胰岛细胞纯度大于90％；胰岛细胞活率大于90％。在低糖和高糖刺激下，胰岛素分泌量分别为（25．42±4．22）mIU·L-1和（38．75±4．56）mIU·L-1，差异具有统计学意义（P〈0．01）。结论胶原酶P逆行灌注消化胰腺、Ficoll 400非连续密度梯度离心法可获得形态、功能完好、高纯度、高活率的胰岛。

Isolation,purification and function test of rat islet

Objective To explore the method obtaining rat islets with enough quantity,higher purity and fine function,and to establish the basis for islet transplantation.Methods Adult rat islets of langerhans were isolated by a collagenase P digestion technique and discontinuous Ficoll density gradient centrifugation method.The changes of cultured islets were observed under a microscope.Islets purity was assessed by dithizone staining.The viability of islets was assayed by fluorescence staining of aridine orange(AO) and propidium iodide(PI).The function of purified islets was determined by glucose stimulated insulin releasing test.Results The total number of purified islets from one rat was(512±89)IEQ.The average purity and viability of islets were more than 90 %,respectively.Insulin secretion of the islets was(38.75±4.56)mIU·L-1 under high glucose stimulation,which was higher than that under low glucose stimulation(25.42±4.22)mIU·L-1(P<0.01).Conclusion The digestion of collagenase P and discontinuous Ficoll density gradient centrifugation can obtain a high yield and high purity islets of pancreas in rats.