人胰岛细胞消化与提纯改良方法研究  

Study of modified methods of digestion and purification of human islet cells

作　　者：谢炬平 蒋小峰[1] 黄镇辉 张大伟[1] 梁浩 薛平[1] Xie Juping;Jiang Xiaofeng;Huang Zhenhui;Zhang Dawei;Liang Hao;Xue Ping(Department of Hepatobiliary Surgery,the Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China)

机构地区：[1]广州医科大学附属第二医院肝胆外科

出　　处：《中华肝脏外科手术学电子杂志》2019年第6期557-560,共4页Chinese Journal of Hepatic Surgery(Electronic Edition)

基　　金：广州市科技计划项目（201607010033）;广州医科大学附属第二医院新技术新业务临床研究与应用项目（Y-03）

摘　　要：目的探讨改良人胰岛细胞消化与提纯方法。方法胰腺供体来源于20例成人公民逝世后器官捐献(DCD)供者捐献,由广州医科大学附属第二医院器官移植中心提供,且通过医院伦理委员会审查。供者男13例,女7例;平均年龄(45±7)岁。4℃的UW液腹主动脉灌注后迅速将胰腺切除,胰腺组织4℃UW液保存,胰管修剪后灌注胶原酶V,放入新型自制改良胰腺消化罐,38℃的胶原酶消化液恒温消化30 min,通过不连续密度梯度法分离提纯胰岛细胞,通过双硫腙(DTZ)染色显示胰岛细胞,计算胰岛当量。吖啶橙(AO)染色法测定存活率。结果共消化20例成人胰腺,胰腺组织消化率为92.7%。单次消化的胶原酶使用量为400 ml。胰岛细胞经过DTZ染色后呈猩红色的类圆形胰岛细胞团。每条胰腺消化后的胰岛细胞当量为(69±28)×10^3 IEQ/g,提纯后为(53±29)×10^3 IEQ/g,纯度为79%。胰岛存活率为(94.1±2.3)%。结论采用我院新型自制改良胰腺消化罐可获得良好的人胰岛素细胞消化和提纯效果,且可明显减少胶原酶的使用量。Objective To explore the modified methods of digestion and purification of human islet cells.Methods Donor pancreases from 20 donors after cardiac death(DCD)were provided by the Organ Transplantation Center of the Second Affiliated Hospital of Guangzhou Medical University and the local ethical committee approval was received.Among 20 donors,13 cases were male and 7 female,aged(45±7)years on average.The pancreas was resected immediately after perfusion of 4℃UW solution in the abdominal aorta and the pancreatic tissues were stored in 4℃UW solution.The trimmed pancreatic duct was injected with collagenase V and was digested with collagenase digestion juice at 38℃for 30 min in a self-designed pancreatic digestion tank.The islet cells were isolated and purified by discontinuous density gradient centrifugation method and were stained with dithizone(DTZ)staining.The islet equivalent quantity was calculated.The survival rate of islet cells was determined by acridine orange(AO)staining.Results A total of 20 samples of adult pancreases were digested with a digestive rate of 92.7%.The amount of collagenase for single digestion was 400 ml.After DTZ staining,the islet cells were observed in scarlet round-like cell clusters.The islet equivalent quantity of each pancreas after digestion was(69±28)×103 IEQ/g,and(53±29)×103 IEQ/g after purification with a purity of 79%.The survival rate of islet was(94.1±2.3)%.Conclusions Excellent digestion and purification effect of human islet cells can be obtained by using the novel self-designed modified pancreatic digestion tank,which can significantly reduce the dosage of collagenase.

关 键 词：胰岛细胞 消化 提纯 移植 

分 类 号：R58[医药卫生—内分泌]