新生期小鼠胰岛分离纯化方法的研究
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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)


Isolation and purification of mice pancreatic islets during neonatal period
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    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< 50 μm胰岛居多(P < 0.01),而3周以50~99 μm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。

    Abstract:

    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 -滋m at 1 w (P < 0.01),and between 50~99 -滋m 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. [Key words] neonatal period;mice;pancreatic islets;isolation and purification

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朱嘉瑜,王晓兢,张勤凤,徐一力,武晓泓.新生期小鼠胰岛分离纯化方法的研究[J].南京医科大学学报(自然科学版),2013,(6):766-770

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  • 收稿日期:2013-04-17
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  • 在线发布日期: 2013-06-25
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