Wistar大鼠胰岛细胞体外分离、纯化及鉴定(英文)

THE ISOLATION,PURIFICATION AND IDENTIFICATION OF WISTAR RAT'S ISLET

目的探索Wistar大鼠胰岛分离纯化的最佳条件。方法采用肝胰管内灌注胶原酶消化分离胰岛及Ficoll 400密度梯度离心纯化。纯化后的胰岛经组织学染色、电镜及放射免疫法鉴定其特异性和活力。结果组织学染色显示纯化后胰岛的活力和纯度分别在95%和85%以上。电镜显示纯化后的胰岛形态完整,包膜清晰,分泌颗粒丰富。放射免疫结果表明,低糖组和高糖组分泌胰岛素的浓度有显著差异,证明胰岛功能良好。结论肝胰管内灌注胶原酶消化法是一种好的消化方法。影响胰岛收获量的因素很多,例如胰腺的充分扩张,胶原酶的浓度和活性以及消化的时间等。

Objective The experiment aims at probing the best condition of the isolation and purification of rat islets. Methods The islets were isolated from rat pancreas by hepatopancreatic duct perfusing with collagenase and purified with Ficoll 400 discontinuous density gradient centrifugation. Then the purified islets were subjected to histological staining, electron microscopy and radioimmunoassay for identification of specificity and viabihty. Results The histological staining revealed that the viability and the purity of the purified islets were above 95% and 85% respectively. Electron microscopy showed that the purified islets were morphologically intact with clear membrane and plenty of secreting granules. Radioimmunoassay demonstrated the secreted insulin concemtration between low-glucose groups and high-glucose groups varied significantly, which verified the good function of the islets. Conclusion Hepatopancreatic duct perfusing with collagenase is a good method for digestion. There are many factors that influence the quantity and quality of the acquired islets, such as the completed expansion of pancreas, the concentration and viabihty of collagenase and the digested time, etc.