中文摘要：

目的：建立一种经济高效的小鼠胰岛细胞分离纯化方法，为进行NOD小鼠的胰岛移植提供实验条件。方法：将5—7周龄、体重20-25g的雄性昆明小鼠的胆总管结扎，并逆行Hank’s液和胶原酶P灌注和分离消化，依次加入84％、67％、50％浓度的Histopaque介质后进行不连续密度梯度离心纯化胰岛细胞。双硫腙（dithizon，DTZ）和台盼兰染色分别鉴定胰岛细胞。用含5．6mmol／L葡萄糖DMEM培养液体外培养胰岛细胞，培养后的第3、5、7、9、11天取细胞上清检测胰岛素水平，并用16．7mmol／L的高浓度葡萄糖进行刺激，检测胰岛素水平确定胰岛细胞功能。结果：每个胰腺的胰岛细胞收获量在1200±124个，且纯度和活性均大于90％；体外培养9天内胰岛细胞基础胰岛素分泌水平无显著差异，至第11天时则明显减少（P〈0．05）；应用16．7mmol／L葡萄糖刺激后，第5、7、9、11天的胰岛素分泌水平较第3天的明显减少（P〈0．05），然而在第7天、9天、11天时的胰岛素水平较第5天时显著降低。结论：胆总管逆行注射Hank’s液和胶原酶P消化消化和不连续密度梯度Histopaque纯化的方法可以获得大量状态良好的胰岛，且胰岛细胞数量多，分泌状态良好。本分离方法是一种经济高效的胰岛细胞分离方法，同时离体后于5．6mmol／L葡萄糖DMEM培养第3天胰岛细胞胰岛素储备功能最佳，为移植研究的最佳状态。

英文摘要：

Objective： To set up an economic and effective method for islet isolation from mice, and prove a laboratory protocol of animal model for clinical islet transplantation. Methods： Adult male Kunming mouse weighing 20-25 g were used as research targets. In each of 5 repeated experiments, pancreatic islets of animals were isolated by infusion of Hank＇s solution, which contained 0.1mg/L collagenase P solution, then the mouse pancreas were digested, Islet purification was performed by using a discontinuous density gradient centrifugation that was prepared with 84%, 67%, 50% of Histopaque. Islet yield and purity were determined by dithizon （DTZ） stain, and trypan blue stain was used to check viability of islets. The endocrine secretory function was assessed by insulin secretion in 5.6 mmol/L glucose after 11 days incubation of culture in DMEM media, and the cells were stimulated by 16.7 mmol/L glucose. Results： Average isolated number of islets was 1200＋ 124. The average purity and viability of islets were over 90% respectively. After different days of culture, insulin secretion of the islets was significantly lower on the eleventh day than that of other days （P〈0.05）. Stimulated by 16.7mmol/L, insulin secretion of the islets on the third day was significantly higher than that of other days （P〈0.05）. Conclusion： The islet isolation with Hank＇s solution and digestion with low concentration of collagenase P decrease experimental cost and also have a beneficial effect on islet recovery and function. And purified islet cells were cultured in 5.6 mmol/L glucose DMEM media on the third day, which had good condition for islet cells transplantion.