小鼠胰岛移植模型的系列改进

Serial Improvements in Mouse Islet Transplantation Models

目的改进小鼠胰岛的分离、纯化及移植方法。方法基于传统小鼠胰岛移植模型,优化胶原酶消化液配方,标准化胰腺组织的消化过程,改用Histopaque 1077作为纯化胰岛的离心介质,改进胰岛制备物输送至肾包膜下的方法;相差显微镜下观察所获胰岛的形态,用双硫腙(DTZ)对胰岛进行特异性染色并计算胰岛产量及纯度;采用吖啶橙-碘化丙啶(AO-PI)染色法观察胰岛活性;制备糖尿病小鼠模型,设计胰岛移植组(10只)及假手术对照组(10只),检验胰岛移植物治疗糖尿病的效果。结果健康胰岛有完整包膜,DTZ染色后呈猩红色,平均每只小鼠可收获(200±30)个胰岛团,终纯度(90±5)%;荧光显微镜下,AO-PI染色后,正常胰岛显示绿色,死亡胰岛显示红色,胰岛存活率为(93±3)%;糖尿病小鼠接受同系胰岛移植后,90%的小鼠逆转糖尿病,对照组小鼠的血糖无改善。结论成功改进胰岛移植模型,简化和标准化小鼠胰岛的分离、纯化及移植过程,有助于该模型成为胰岛移植研究的常规方法。

Objective To modify the methods of isolation and purification of islets and the islet delivery technique for islet transplantation in a mouse model.Methods Based on the traditional methods,the collagenase digestion solution was adjusted and the standard process of pancreatic digestion was obtained.Histopaque 1077 was adopted as a centrifuge medium for islet purification,and the technique of islet delivery to the subcapsule of the kidney was modified for transplantation.The morphology of islets was observed under the phase contrast microscope,the number and purity of the islets were examined by Dithizone staining,and the vitality of the islets was analyzed by AO-PI immunofluorescence staining.Moreover,the diabetic mice were prepared and divided into the islet transplantation group（n=10）and the sham group（n=10）.The efficacy of the islet transplants in the treatment of diabetics was evaluated.Results The healthy islets had integrated outer membrane.An average of（200± 30）islets was generated per pancreas by the modified method with a final purity of（90±5）%.The AO-PI immunofluorescence staining revealed that the normal islets had green fluorescence,the dead ones were in red,and the vitality of the islets reached（93±3）%.After islet transplantation,the serum glucose level of the diabetic mice recovered to normal in 90%recipients,while no change was observed in the mice of the sham group.Conclusion The islet transplantation model was successfully modified.The isolation,purification and transplantation of islets were simplified and standardized.This model holds promises to be the routine method for islet transplantation research.