小鼠胰岛分离纯化方法研究

Research on isolation and purification method of pancreatic islets in mice

目的探讨可提高胰岛产量和功能活性的小鼠胰岛分离、纯化的方法。方法通过胆总管逆行灌注胶原酶溶液消化小鼠胰腺,经不连续密度梯度离心后,人工挑取、纯化胰岛。过夜培养后,用葡萄糖刺激胰岛素分泌实验(GSIS),检测胰岛功能。电子显微镜(电镜)观察胰岛β细胞亚细胞结构变化。结果利用该改良方法平均每只小鼠可收集(200±20)个胰岛,胰岛直径大小为(175±22)μm。GSIS结果发现胰岛素水平在低糖(2.8 mmol/L)和高糖(16.7 mmol/L)刺激下分别为(0.33±0.07)、(1.36±0.47)ng/(islet·60 min),高糖刺激的胰岛素水平是低糖刺激的4.12倍,差异有统计学意义(P<0.05)。电镜证实胰岛β细胞胞膜、线粒体膜完整,胞内可见大小不一的胰岛素分泌泡。结论胆总管逆行灌注胶原酶消化小鼠胰腺,体外不连续密度梯度离心联合人工挑取的方法是一种简便、快捷、稳定的小鼠胰岛分离方法,小鼠胰岛产量较高、形态完整,且GSIS反应性良好。

Objective To investigate a method for isolation and purification of pancreatic islets in mice, aiming to enhance the quantity and activity of pancreatic islets. Methods The pancreas were digested by retrograde common bile duct perfusion of collagenase, discontinuous density gradient centrifugation and the islets were selected and purified by manual method. After overnight culture, the pancreatic islets were incubated by static glucose-stimulated insulin secretion （GSIS） and the islet function was assessed. The subcellular structure of islet 13 ceils was observed under transmission electron microscopy. Results Using the modified method, （200 ＋ 20） islets were collected in each mouse with a mean diameter of （175 ± 22） μm. The insulin levels in the stimulation of low （2. 8 mmol/L） and high glucose （16. 7 mmol/L） were （0. 33± 0.07） and （1.36 ±0. 47 ） ng/ （islet · 60 min）, which were detected by GSIS. Insulin levels in the stimulation of high sugar is 4. 12 times of those of low sugar with a statistical significance （P 〈 0. 05） . It was revealed by transmission electron microscopy that the pancreatic β cell membrane and mitochondrial membrane was intact and insulin granules of different sizes could be seen within β cells. Conclusions Retrograde common bile duct perfusion of collagenase, discontinuous density gradient centrifugation combined with manual selection in vitro is a convenient, fast and stable method for isolating mouse islets, which can get pancreatic islets with relatively high output, intact cellular morphology, and good reactivity of GSIS.