成年猪胰岛分离方法的改进

Improvement of islet isolation of adult porcine pancreas

目的 :开发成年猪胰岛分离方法。方法 :取成年猪胰腺体尾部 ,胰管内注入冷 0 .1 %胶原酶消化液 ,在 38.5℃水浴箱中消化后 ,放入 4℃ Hanks液中分离并用 60 0μm的滤过网过滤回收。残留的组织重新悬浮在冷 Hanks液中 ,并放入 Ricordi′s chamber内振荡 5min后再次过滤回收。用Ficoll不连续密度液梯度离心法进行胰岛纯化。结果 :纯化前、后胰岛收获量分别为 ( 850 5± 3349)g-1〔IEQ:( 4 2 53± 2 732 ) g-1)、( 2 334± 1 2 2 3) g-1〕,纯度 >80 %。胰岛收获量与消化后的消化液胰蛋白酶活性显示了弱的负相关 ( r=- 0 .44,P=0 .1 3) ,胰岛收获量≥ 80 0 0 g-1组的消化后消化液胰蛋白酶活性明显低于胰岛收获量 <80 0 0 g-1( 78.3± 2 6.7vs1 37.5± 48.4BAEEU,P<0 .0 5)。结论 :我们采用的胰岛分离方法能够获得大量的猪胰岛细胞 ;胰蛋白酶活性影响胰岛收获量。

Objective:To develop the method of adult porcine pancreas islet isolation.Methods:The tails of adult porcine pancreases were perfused through the pancreatic duct with 0.1% cold collagenase solution and incubated as 38.5℃.The digested glands were dispersed in 4℃ hanks balanced salt solution (HBSS).The tissue suspensions were filtered through a 600 μm mesh.The residual tissue was resuspended in cold HBSS,put in the Ricordi′s chamber and shaken for 5 minutes,then filtered again.Purification was achieved by the Ficoll gradient technique.Results:Before purification,the number of islet yields and islet equivalents (150 μm diameter islets) was 8 505±3 349 islets/g pancreas and 4 253±2 732 islets/g pancreas,respectively.After purification,the number of recoveries was 2 334±1 223 islets/g pancreas.The rate of islet purity was over 80%.The islet yield showed negative correlation to the trypsin activity of the solution after incubation (r=-0.44,P=0.13).The trypsin activity in case of higher islet yield (≥8 000 islet/g pancreas) was significantly lower than that of less islet yield (<8 000) in the solution after incubation (137.5±48.4 vs.78.3±26.7 BAEEU,P<0 05).Conclusion:The method can obtain a high yield;trypsin activity can affect islet yield. 〔