半自动纯化法分离纯化成年猪胰岛细胞的实验研究

Semi-automatic Isolation and Purification of Adult Pig Islet Cells

目的探索大规模猪胰岛细胞分离纯化的方法.方法采用胶原酶消化法和Ficoll液不连续密度梯度离心进行分离纯化;采用双硫腙染色、AO-PI染色、胰岛素释放试验检测胰岛的纯度和活性.结果消化后平均获取的胰岛细胞团数量为(4 387±617)IEQ/g胰腺组织;纯化后平均为(3 192±756)IEQ/g胰腺组织;获取的猪胰岛细胞平均纯度为(60.15±2.35)%,活率为(80.00±0.47)%.结论所获得的胰岛细胞有较好的纯度和良好的生物活性,显示半自动纯化法分离纯化成年云南小耳猪胰岛细胞是成功可行的,可以满足进一步异种移植的研究需要,可为大规模分离纯化猪胰岛细胞提供技术支持.

Objective To establish effective method for large-scale purification of islet cells from pig pancreas.Methods Pig pancreas tissue was digested with collagenase P through continuous infusion.Pig islets was purified by Ficoll discontinuous density gradient centrifugation medium.Dithizone（DTZ）staining was used to observe the morphology of islet cells clusters（ICCS）,to count islet equivalent IEQ,and to detect the purity of the islet.AO-PI staining was used to detect islet viability and the islet function assessed by insulin release assay in vitro.Results The number of the islets collected was（4 387±617）IEQ/g in average from pancreas before purification,and（3 192±756）IEQ/g pancreas after purification.The average purity of islets cells was（60.15±2.35）%,and the motilityrate was（80.00 ± 0.47）%.Conclusions The islet cells obtained by this method have better purity and good biological activity.That＇s to say the Semi-automatic isolation and purification of small-eared pig islet cells is successful and feasible.The established method can be applied in large-scale purification of fully functional islet cells from pig pancreas,and can meet the need for further studies of xenotransplantation.