猪胰岛的分离与纯化

Isolation and purification of pig islet cells

目的探索大规模猪胰岛细胞分离纯化的方法。方法联合器官切取，胶原酶P主胰管灌注，COBE2991细胞分离机及HCA-Ficoll纯化猪胰岛细胞。通过双硫腙（DTZ）染色，倒置显微镜下计数胰岛细胞的数量和纯度，胰岛素释放试验检测胰岛细胞的分泌功能。结果消化后平均每条胰腺可平均获得（275000±20895）胰岛细胞当量（IEQ），纯化后平均为（230350±26679）IEQ，平均每克胰腺组织可获得（2710±229）IEQ，纯化后胰岛细胞平均纯度为（50．2±1．95）％。纯化后的胰岛细胞对胰岛素释放刺激反应良好，高糖（16．7mmol／L）时胰岛素的释放量为低糖（3．3mmoL／L）时的4．74倍（P≤0．001）。结论成功建立了猪胰岛细胞分离、纯化的方法，纯化的猪胰岛细胞具有良好的生物活性。

Objective To establish effective method for large-scale purification of islet cells from pig pancreas. Methods Pig pancreas tissue was digested with collagenase P followed by purification in a HCA-Ficoll dis continuous gradient using Cobe2991 cell separator. After isolation, the islet cell yield and purity were evaluated with light microscope with DTZ staining, and the islet function assessed by insulin release assay in vitro. Results The number of the islets coll ected from each pancreas averaged （275 000 ± 20 895 ） islet equivalents （IEQ） before purification, and （230 350 ± 26 679） IEQ after the purification with discontinuous gradient eentrifugatian. From each gram of the pancreatic tissue, （2710 ±229） IEQ were obtained with an average purity of （ 50. 2 ± 1.95 ） %. The purified islets responded well to high-concentration （16.7 mmol/L） glucose stimulation with a 4. 74-fold increase of insulin secretion over the basal level （3.3 mmol/L, P 〈 0.001 ）. Conclusion The established method can be applicable for large-scale purification of fully functional islet cells from pig pancreas.