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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 investigate the isolation and purification of pancreatic islets in neonatal mice. Methods: The pancreatic islet filling and collagenase Ⅴ digestion were used to compare the effects of different digestion time and purification of Histopaque-1077 on pancreatic islets under the microscope. The effects of insulin and glucagon double staining were detected by flow cytometry Islet β and α cell ratio. RESULTS: At 1 mg / ml enzyme concentration, the optimum digestion time was 23 min (islets / pancreas 38.11 ± 2.78) and 25 min (islets / pancreas 66.06 ± 2.61) at 1 week and 3 weeks, respectively. The number of islets in one week was mostly less than 50μm (P <0.01), while the number of islets was mostly in the range of 50 ~ 99μm in three weeks (P <0.01). The yield of islets in the hand-pick group was significantly higher than that in the Histopaque-1077 group (P <0.05). The viability and purity of the obtained islets were> 90%, and there was a dynamic change in the proportions of β and α cells (P <0.05). CONCLUSION: Pancreas is partially filled and digested with collagenase Ⅴ. It is a good method for pancreatic islet isolation and purification in neonatal mice. Digestion time and purification methods are important factors.