摘要
目的:探讨获得高质量小鼠胰岛的分离纯化方法,评价其功能。方法:采用胆总管内胶原酶灌注膨胀消化胰腺的方法分离小鼠胰岛,不连续密度梯度离心法纯化胰岛,用双硫腙(Dithizone,DTZ)对胰岛进行特异性染色计算胰岛产量及纯度,以葡萄糖和茶碱刺激胰岛素释放检测胰岛功能。结果:胰岛的产量和活性主要与胰腺均匀膨胀和胶原酶的消化时间有关。平均每个小鼠胰腺能得到150~250个高质量胰岛,活性>95%,纯度>90%。葡萄糖及茶碱(carbachol,Cch)刺激后胰岛素释放量明显增加。结论:改良的胆总管内胶原酶灌注膨胀消化小鼠胰腺及不连续密度梯度Ficoll-400纯化胰岛的方法,可获得产量较高、纯度及功能较好的胰岛。
Objective: The technical difficulty in mouse islets isolation and purification restricts its wide use in diabetes and islet transplantation field. Here we reported and discussed a modified method with which to isolate and purify high quality islets in mice. Methods:Intraductal collage- nase-P perfusion and uniform distention of the gland were applied to digest mice pancreas. A Ficoll gradient solution (25%, 23%, 20%, and 11%) was applied to make the purification. Islets were then tested for their specificity by Dithiozine (DTZ) staining. The islet function was assessed by the glucose- and carbachol-stimulated insulin secretion test. Results: The yield and viability of the islets were closely associated with good gland distention and collagenase digestion timing. We used the method discussed here, each mouse pancreas could yield 150-250 islets, with viability 〉95% and purity 〉90%. The insulin release from the in vitro islets fed with high concentration glucose and Carbachol stimuli raised to a significantly higher level. Conclusion:The above-mentioned method of mice isolation and purification might yield high quality islets for further scientific use.
出处
《中国现代普通外科进展》
CAS
2007年第5期378-381,共4页
Chinese Journal of Current Advances in General Surgery
基金
2005年度上海市科委"青年科技启明星"基金资助项目(05QMX144)