NOD小鼠胰岛分离与纯化的方法研究

Isolation and purification of islets in non-obese diabetic mice

目的 :研究非肥胖性糖尿病 (NOD)小鼠胰岛分离与纯化的方法。方法 :采用改良的胰导管逆行灌注胶原酶静止消化胰腺 ,分离成年NOD小鼠胰岛 ,不连续密度梯度Ficoll液 (2 5 % ,2 3% ,2 0 5 % ,11% )离心、纯化胰岛。用双硫棕 (Dithizone ,DTZ)对胰岛进行特异性染色 ,以葡萄糖刺激胰岛素释放检测胰岛功能。结果 :不同浓度 (0 5mg·ml-1,1mg·ml-1,2mg·ml-1)的胶原酶在不同时间 (2 0min ,4 0min ,6 0min)内静止消化胰腺后收获的胰岛数量和胰岛活性不同 ,采用 1mg·ml-1浓度的Ⅺ型胶原酶消化 4 0min后效果最好 ,平均能得到 (195± 8)个胰岛 /胰腺 ,活性 >90 % ,纯度 >90 %。DTZ染色后胰岛呈红色 ,形态完整。高糖刺激后胰岛素释放量为低糖刺激后的 2倍。结论 :改良的胰导管逆行灌注胶原酶静止消化NOD鼠胰腺 ,及不连续密度梯度Ficoll液纯化胰岛的方法 ,可获得数量较多 ,纯度较高和功能较好的胰岛。

Objective To investigate the method of isolating and purifying islets in non obese diabetic(NOD) mice. Methods The isolation of adult NOD mice pancreatic islets was carried out by stationary digestion after pancreatic ductal injection of collagenase solution. A discontinuous Ficoll solution(25%,23%,20.5%,11%) was applied to the purification of the islets. The specificity of the islets was determined by dithizone(DTZ) staining, and the function was evaluated by the glucose stimulating insulin releasing test. Results The yield and activity of the islets varied with different concentration collagenase and digesting times. After incubation of the pancreas with 1.0 mg·ml -1 Type Ⅺ collagenase for 40 min, each pancreas could yield (195±8) islets, with viability>90% and purity>90%. The islets were red and intact after DTZ staining. The insulin from the islets fed with high concentration glucose was 2 times that with low concentration. Conclusion The modified method of pancreatic ductal collagenase injection, followed by stationary digestion and purification of islets with a discontinuous Ficoll solution, can reproducibly yield various and pure islets.