大鼠胰岛的分离、纯化与评价方法改良  

Isolation,purification and assessment of rat pancreas islet

作　　者：曾明华[1] 陈树林[1] 蒋田园[1] 毛海鹏[1] 蒲强红[1] 周建平[1] 李耀辉[1] 林磊[1] 李文献 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]河南省乳品工程技术研究中心,河南郑州450008

出　　处：《西北农林科技大学学报（自然科学版）》2008年第2期50-54,60,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(30170683);陕西省自然科学基金项目(2006C130);河南省乳品工程技术研究中心科研基金项目(2006001)

摘　　要：【目的】简化胰岛的分离纯化方法,建立完善的胰岛评价体系。【方法】采用Ⅴ型胶原酶分离出Spra-gue-Dawley大鼠胰岛,并采用改良的3层Dextran不连续密度梯度离心法纯化胰岛。纯化的胰岛通过双硫腙(Dithi-zone,DTZ)染色进行计数及纯度检测,丫啶橙(AO)-碘化丙啶(PI)双染色法确定分离细胞的存活率,体外葡萄糖刺激释放胰岛素试验检测胰岛的分泌活性。采用Adobe Photoshop程序改进了常规的胰岛计数方法,并进行胰岛标准计数。【结果】平均每只成年Sprague Dawley大鼠的胰腺,可获得(3 840±24)当量数(IEQ)纯化胰岛,纯度可达到90%,细胞平均存活率达92%。【结论】采用改良的3层Dextran不连续密度梯度离心法及基于Adobe Photoshop程序的标准计数方法,可分别用于大鼠胰岛分离纯化和计数。[Objective] This study was conducted to simplify the method of islet isolation and to establish intact islets evaluation system. [Method] Adult rat pancreatic islets were isolated by type V collagenase digestion and purified by Dextran discontinuous density gradient centrifugation. Counting and purity detection of the purified islets were performed under stereoscopic microscopes after staining with Dithizone. The viability of the cultured islets was determined by acridine orange-propidium iodide double-staining. Insulin secretory activity of the islets was detected by glucose challenge tests. A standard islet counting was conducted via Photoshop program by the improved method. [Result] There were on average （3 840±24） IEQ purified islets isolated from the pancreas of one Sprague Dawley rats. The purity of islets could reach 90%. And the average viability was as high as 92%. [Conclusion] The islets purified by three- layer Dextran density gradient centrifugation still kept intact structure and functional integrity and were relatively higher in quantity. The counting method based on the Photoshop program simplified the procedures and was more accurate.

关 键 词：大鼠胰岛 分离纯化 胰岛计数 分泌活性 

分 类 号：R587[医药卫生—内分泌] S865.12[医药卫生—内科学]