新生期小鼠胰岛分离纯化方法的研究

发布时间：2019-05-16 18:45

【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(P0.01),而3周以50～99μm者居多(P0.01)。单纯手挑组胰岛得率显著高于Histopaque-1077纯化组(P0.05)。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(P0.05)。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 explore the isolation and purification scheme of neonatal mouse islets. Methods: pancreatic local filling and collagenase V digestion were used to compare the effects of different digestion time and simple microscope selection or Histopaque-1077 purification on islet yield. The proportion of islet 尾 and 伪 cells was detected by flow cytometry after insulin / glucagon double staining. Results: at the concentration of 1 mg/ml, The optimum digestion time of islets in 1 week and 3 weeks was 23 min (islet / pancreas 38.11 卤2.78) and 25 min (islet / pancreas 66.06 卤2.61), respectively. In the third week, 50 ~ 99 渭 m was the most common (P 0.01). The islet yield in the simple hand selection group was significantly higher than that in the Histopaque-1077 purified group (P 0.05). The survival rate and purity of islets were 90%, and there were dynamic changes in the proportion of 尾 and 伪 cells (P 0.05). Conclusion: local filling of pancreas and endoscopic selection of islets after digestion of collagenase V is a good method for isolation and purification of islet in neonatal mice, and digestion time and purification method are important influencing factors.
【作者单位】：南京医科大学第一附属医院内分泌科;
【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)
【分类号】：R-331

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