大鼠胰岛分离及纯化方案的改进  

Improved method for rat pancreatic islets isolation and purification

作　　者：肖芳[1,2,3] 李文娟[1,2,3] 侯新国[1,2] 宋君[1,2] 校娟[1,2] 王美建[1,2] 刘继东[1,2] 邓楠[1,2] 周晓莹[1,2] 陈丽[1,2] 

机构地区：[1]山东大学内分泌代谢病研究所,济南250012 [2]山东大学齐鲁医院内分泌科,济南250012 [3]山东大学细胞治疗研究中心,济南250012

出　　处：《山东大学学报（医学版）》2012年第3期8-10,16,共4页Journal of Shandong University:Health Sciences

基　　金："十一五"国家科技支撑计划项目(2006BAI02B08;2009BAI80B04);山东省自然科学基金资助项目(Z2008C05);山东省科技攻关国际科技合作项目(2010GHZ20201);山东大学自主创新基金(2009TS054);济南市科技局科技明星项目(20100318)

摘　　要：目的优化大鼠胰岛分离纯化条件,为糖尿病胰岛受损的发病机制研究及胰岛再生的应用提供必要材料。方法胆总管原位灌注胶原酶V消化胰腺并分离胰岛后,比较经典不连续密度梯度Ficoll离心法和Histopaque1077密度离心法纯化胰岛的效果,其间观察胰腺灌注液体积及消化时间不同对大鼠胰岛分离纯化的影响,并对纯化后的胰岛进行数量、纯度和功能的体外检测。结果 Histopaque 1077密度离心法比经典不连续密度梯度Ficoll离心法可得到更多优质的胰岛(P<0.01)。两种方法获得的胰岛在高糖刺激下释放胰岛素的能力差异无统计学意义(P>0.05)。胰腺灌注液之体积及消化时间对胰岛提取有十分重要的影响。结论 Histopaque 1077密度离心法简单易行且效率较高,有望成为纯化大鼠胰岛的首选方法,便于临床和基础研究工作的开展。Objective To optimize conditions for rat pancreatic islets isolation and purification,and provide experimental data for research on the pathogenesis of islet injury in diabetes and application of islet regeneration.Methods Influences of different pancreatic perfusion volumes and digestion time on rat pancreatic islets isolation through intraductal collagenase V perfusion in situ were observed.Effects on islets purification were compared between classical discontinuous Ficoll density gradient centrifugation and Histopaque 1077 density gradient centrifugation.Afterwards,the islets were examined and their morphology,quantity,purity and function were evaluated in vitro.Results Histopaque 1077 density gradient centrifugation could yield more islets of high quality（P0.01）.Insulin release by the islets through the two different methods under high glucose condition has no differentiation（P0.05）.Islets perfusion volumes and digestion time played significant roles in rat islets extraction.Conclusion Histopaque 1077 density gradient centrifugation,with simple operation and high yield,has a prospect to become the most effective method for rat islets purification and it will be more useful in clinical and basic research in the future.

关 键 词：大鼠胰岛 分离 纯化 胶原酶V 

分 类 号：R587.1[医药卫生—内分泌]