SD大鼠原代胰岛细胞分离、纯化及培养技术的改进

Isolation,Purification and Culture of Islet Cells from SD Rat

目的：探讨大鼠胰岛细胞分离、纯化及培养的方法，并评价其生物学功能。方法：选用8-10周龄健康SD大鼠，采用胆总管逆行注射预冷胶原酶P溶液，37℃水浴静止消化，30目不锈钢筛网过滤，Ficoll400非连续密度梯度离心纯化。分离后的胰岛用DTZ染色计算胰岛产量，胰岛素释放试验评价其生物学功能。结果：胰岛细胞分布于Ficoll400浓度为23％～20％和20％～11％的界面之间。DTZ染色呈红色细胞团，胰岛产量为（606±56）IEQ／胰腺。纯度高达80-90％，活率≥90％，胰岛素释放功能良好。结论：胶原酶P溶液原位消化，Ficoll400纯化是一种高效简便的胰岛分离方法，分离的胰岛细胞数量多、纯度高及活性好。

Objective： To establish a method of isolation, purification and culture of rat islet cells and evaluate their biological characteristics. Methods： 8-10 week-old healthy adult SD rats were selected. Pre-cooling duct retrograde injection of collagenase P solution, 37℃ water bath stationary digesting, 30 mesh stainless steel screen filter and Ficoll400 discontinuous density gradient centrifugation were used for isolation and purification. The purified cells were detected for islet equivalent by DTZ staining, and their biological function was evaluated by insulin release test. Results： Islets distributed in the interfaces between Ficoll400 concentration of 23% and 20% as well as between 20% and 11%. DTZ staining showed red cell mass; islet yield was （606 ± 56） IEQ / pancreas with up to 80-90% purity; viability was - 90% and the islet cells showed well release of insulin. Conclusion： In situ digestion by collagenase P followed by Ficoll400 purification is an efficient and easy method of islet isolation, yielding cells of large quantity, high purity and good activity.