叙利亚金黄地鼠胰岛的分离、纯化与功能鉴定

Isolation, purification and functional identification of Syrian golden hamster islets

目的构建成年叙利亚金黄地鼠胰岛的分离、纯化及功能鉴定的方法。方法胶原酶V灌注分离成年叙利亚金黄地鼠胰腺,不连续密度梯度法纯化胰岛。经DTZ染色后于倒置显微镜下测定胰岛细胞的数量和纯度。通过胰岛素释放试验检测胰岛细胞的功能。结果纯化后每只地鼠胰腺可获得(359±35)胰岛细胞当量,胰岛纯度>90%,胰岛活率>90%。在低糖和高糖刺激下,胰岛细胞体外培养液中胰岛素含量的浓度分别为(3.29±0.32)mU/L和(11.12±0.57)mU/L。高糖刺激后胰岛素释放量为低糖的3.38倍。体外培养1周生长状况良好。结论成功获取了高纯度、高活率的金黄地鼠胰岛细胞。

Objective To establish the methods for isolation, purification and function identification of Syrian golden hamster islets. Methods The Langerhans islets were isolated and purified from golden hamster pancreas by intra-ductal collagenase V perfusion and discontinuous Ficoll density gradient centrifugation. After isolation, the islet yield and purity were evaluated with DTZ staining. The islet function was assessed using glucose-stimulated insulin secretion test. Results The total number of purified islets from one donor hamster was 359±35 islet equivalent （IEQ）, with the purity and viability of the isolated islets of more than 90%. In response to glucose stimulations at 5.8 and 16.7 mmol/L, the secretion of insulin by the islets was 3.29±0.3 and 11.12 ±0.57 mU/L, respectively, showing a 2.28-fold higher insulin release by high-concentration than by low- concentration glucose stimulation （P〈0.01）. Conclusion The methods of collagenase V digestion and gradient centrifugation result in high yield and high purity of the isolated hamster islets.