摘要
目的:探讨新生期小鼠胰岛分离纯化方案。方法:采用胰腺局部充盈、胶原酶Ⅴ消化,对比不同消化时间及单纯显微镜下手挑胰岛或Histopaque-1077纯化对胰岛得率的影响,并通过胰岛素/胰高血糖素双染后流式细胞术检测胰岛β和α细胞构成比例。结果:在1 mg/ml酶浓度下,1周和3周小鼠胰岛的最适消化时间分别为23 min(胰岛/胰腺38.11±2.78)和25 min(胰岛/胰腺66.06±2.61)。1周以直径<50μm胰岛居多(P<0.01),而3周以50～99μm者居多(P<0.01)。单纯手挑组胰岛得率显著高于Histopaque-1077纯化组(P<0.05)。所得胰岛存活率和纯度均>90%,且存在β和α细胞构成比例的动态变化(P<0.05)。结论:胰腺局部充盈、胶原酶Ⅴ消化后镜下手挑胰岛是一种较好的新生期小鼠胰岛分离纯化方法,消化时间和纯化方法是其重要影响因素。
Objective：To establish a method for the isolation and purification of mice pancreatic islets during neonatal period.Methods：Pancreases of mice at neonatal stage were injected and digested by collagenase Ⅴ.The yield of islets was compared among different digestion time and hand-picked under microscope or Histopaque-1077 purification.The islets were stained with anti-glucagon antibody and anti-insulin antibody,and then identified by flow cytometry.Results：The optimal digestion time of islets from 1-week and 3-week old mice was 23 min（38.11 ± 2.78/pancreas） and 25 min（66.06 ± 2.61/pancreas）.The diameters of most islets were less than 50 μm at 1 w（P〈0.01）,and between 50～99 μm at 3 w（P〈0.01）.The yield of islets purified by hand-picked under microscope was much higher than that of Histopaque-1077（P〈0.05）.The purity and survival rate of islets were above 90%,and the ratio of β and α cells within mice islets changed during neonatal period（P〈0.05）.Conclusion：Local injection and digestion of pancreas with collagenase Ⅴ and hand-picked under microscope is an effective method for mice islet isolation during neonatal period,and the digestion time and purification methods are important.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2013年第6期766-770,共5页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金资助(30971404
81070621
81261120566)
江苏省国际科技合作计划(BZ2011042)
江苏省医学重点人才项目(RC2011068)
江苏省"六大人才高峰"项目(苏人社发【2011】582号)
关键词
新生期
小鼠
胰岛
分离纯化
neonatal period
mice
pancreatic islets
isolation and purification
