一种改良的小鼠胰岛分离方法  

A simple method to isolate mouse islets

作　　者：李伟[1] 徐伟[1] 陈娟[1] 王晓航 孙子林[1] Li Wei Xu Wei Chen Juan Wang Xiaohang Sun Zilin.(Department of Endocrinology, Zhongda Hospital, Institute of Diabetes, Medical School, Southeast University, Nanjing 210009, China)

机构地区：[1]东南大学附属中大医院内分泌科东南大学医学院糖尿病研究所,南京210009

出　　处：《中华糖尿病杂志》2017年第9期561-566,共6页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金（81370874）

摘　　要：目的探讨一种简单快捷、廉价高效的小鼠胰岛分离与纯化方法。方法采用自制的穿刺针由胆总管逆行穿刺并灌注胶原酶Ⅴ消化胰腺，使用仅一种密度梯度介质离心结合手工挑选的方法分离纯化6~8周龄雄性ICR小鼠胰岛。同时，以单纯手工挑选法纯化胰岛作为对照组。双硫腙染色鉴定获取胰岛纯度，吖啶橙/碘化丙啶染色鉴定胰岛活力。采用不同浓度葡萄糖（低糖2.8 mmol/L，高糖20.0 mmol/L）刺激的胰岛素释放实验检测胰岛功能。采用t检验比较两组间的差异。结果采用自制穿刺针及密度梯度离心的新方法提取5只小鼠胰岛整体耗时152 min，而单纯手工挑选法则耗时347 min；新方法每只小鼠平均可得胰岛（95±8）个，略低于单纯手工挑选组的（124±10）个（t=4.027，P=0.016）；但新方法所得胰岛纯度为92.6%±1.3%，显著高于单纯手工挑选法79.2%±5.5%（t=4.075，P=0.015）。同时新方法所提取的胰岛活力与手工挑选法获得的胰岛的活力相当。新方法获得的胰岛在低糖和高糖刺激下上清中胰岛素分泌量分别为（66±13）和（256±46） μg/L（t=7.042，P=0.002），提示这种新方法获取的胰岛对糖的反应很灵敏。结论采用自制的穿刺针及密度梯度离心的方法所得胰岛产量、纯度、活力及胰岛功能都相对良好，是一种简单快捷、廉价高效的小鼠胰岛分离方法。ObjectiveTo develop a simple, cheap and effective method to isolate and purify islets in mouse.MethodsCollagenase V was perfused into the pancreases by a self-made puncture needle inserted into the common bile duct, after digestion islets were isolated by a combined way of density gradient centrifugation and hand-pick. While the islets purified just by hand-pick was treated as control. Dithizone （DTZ） staining was applied to test the purity of islets, while the viability of islets was identified by Acridine orange/iodide c organism （AO/PI） staining and the islet function was tested by glucose（low glucose 2.8 mmol/L, high glucose 20.0 mmol/L） stimulated insulin secretion. Differences between the two groups were analyzed by student-t test.ResultsIt took 152 minutes to harvest the islets from five mice with self-made puncture needle and density gradient centrifugation, and it took 347 minutes with the traditional hand-pick method. The production of islets from each mouse using this new method was 95±8 and was slightly lower than that in control group （124±10, t=4.027, P=0.016）; the purity with the new method was much higher than that in control group （92.6%±1.3% vs 79.2%±5.5%, t=4.075, P=0.015）. There was no notable differences in islets viability between the two methods. Insulin secretion of the islets isolated by this combined method at low glucose （2.8 mmol/L） and high glucose （20.0 mmol/L） environment were （66±13） and （256±46） μg/L, separately （t=4.075, P=0.002）, it indicated that the islets isolated by this new method were sensitive for the change of glucose concentration.ConclusionsThe islets isolated by the combined way of density gradient centrifugation and hand-pick using self-made puncture needle maintain the production, purity, viability and islet function. In general, this method is a simple, cheap and effective way to isolate mouse islets.

关 键 词：糖尿病 胰岛 小鼠 分离纯化 

分 类 号：R285.5[医药卫生—中药学]