简便快捷的小鼠胰岛分离纯化方法研究

目的 探讨小鼠胰岛分离与纯化的方法.方法 采用多点注射灌注胶原酶法消化胰腺,不连续密度梯度离心联合人工挑取的方法分离、纯化胰岛.双硫腙特异性染色后计算胰岛产量及纯度.葡萄糖刺激后测定培养上清液胰岛素水平检测胰岛功能.结果 (1)每只小鼠采用上述分离、纯化法平均得到(114±15)个胰岛,平均纯度为(77.12±3.23)％,胰岛细胞存活率＞90％； (2)分离、纯化的胰岛培养上清液中胰岛素水平在无糖、低糖(2.8 mmol/L)和高糖(22.2 mmol/L)刺激下分别为(23.80±3.52) mIU/L、 (67.57 ±4.04) mIU/L和(164.32±10.75) mIU/L,各组间比较,差异有统计学意义(P＜0.05).两两比较,低糖组的胰岛素水平为无糖组的2.84倍(P＜ 0.05),高糖组的胰岛素水平为低糖组的2.43倍(P＜0.05),高糖组的胰岛素水平为无糖组的6.90倍(P＜0.05).结论 采用多点注射灌注胶原酶法消化胰腺,不连续密度梯度离心联合人工挑取的方法分离、纯化的小鼠胰岛产量及纯度较高,形态完整,不同浓度葡萄糖刺激后胰岛素分泌反应良好,是一种简便、快捷的小鼠胰岛分离方法.

A Convenient and Efficient Method of Mouse Pancreatic Islet Isolation and Purification

Objective To investigate a convenient and efficient method of mouse pancreatic islets isolation and purification.Methods The isolation of mouse pancreatic islets was carried out by stationary digestion after multi-point injection of collagenase Ⅳ solution into the pancreas.A discontinuous Histopaque solution(1119,1110,1080 and 1060) was applied for the purification of the islets.Islet cell purity and viability were determined by dithizone(DTZ) and Trypan blue staining.The islets were cultured overnight and stimulated with different concentration of glucose(0,2.8 and 22.2mmol/L) for 1 hour.Supernatants were collected for insulin assays by an insulin RIA kit.Results(1) By this optimized islets isolation and purification protocol,each mouse pancreas could yield(114 ± 15) islets,with an average purity of(77.12±3.23) % and viability more than 90%;(2) The insulin level in 0,2.8 and 22.2 mmol/L glucose stimulation groups were(23.80±3.52) mIU/L,(67.57± 4.04) mIU/L and(164.32±10.75) mIU/L,respectively.There was a statistically significant difference among groups(P <0.05).Compared with 0 mmol/L glucose group,the insulin level in 2.8 mmol/L glucose group was 2.84 times higher(P <0.05) and the insulin level in 22.2 mmol/L glucose group was 6.90 times higher(P< 0.05).Compared with 2.8 mmol/L glucose group,the insulin level in 22.2 mmol/L glucose group was 2.43 times