试管法纯化人胰岛细胞的初步研究

Preliminary study on the purification of human islet cells by tube method

目的 探索用试管法初步建立纯化人胰岛细胞的方法。方法 用胶原酶P两步法消化分离胰腺 ,用试管法通过不连续密度梯度HC -A -Ficoll纯化液纯化人胰岛细胞。通过DTZ染色 ,在倒置显微镜下计数胰岛细胞的数量和纯度 ,用胰岛素释放试验检测胰岛细胞的功能。结果 纯化后平均每个胰腺可获得 (5 35 0 0±2 1 4 6 5 )个胰岛细胞团 ,平均每克胰腺组织可获得胰岛细胞团 (75 0± 2 6 7) ,平均纯度为 72 92 %。纯化后的胰岛细胞对胰岛素释放刺激反应良好 ,高糖时胰岛素的释放量为低糖时的 1 89倍 (P <0 0 1 )。结论 建立了人胰岛细胞纯化的方法 。

Objective To establish an effective method for purification of islet cells from human pancreata by tube method. Methods Pancreata was enzymatically digested by two-step digestion method and purified by discontinuous gradient centrifugation on four layers of HC-A-Ficoll solutions by tube method. After isolation, the islet yield and purity were evaluated with the light microscope by staining with DTZ. Islet function was assessed by insulin release assays in vitro . Results After the purification step by discontinuous gradient centrifugation, ( 53 500 ± 21 465 ) islets per pancreas and (750±267) islets per gram of pancreas were obtained from human pancreata, with an average purity of 72 92%. The purified islets were respond to high concentration glucose stimulation (300 mg/dl) with 1 89 times increase of insulin secretion over basal levels (60 mg/dl), with significant difference ( P <0 05). Conclusion A method for purification of islet cells from human pancreata was established, and the purified islet cells remain full function.