恒河猴胰岛的半自动分离和纯化

Isolation and purification of islet cells from rhesus monkeys

目的探讨恒河猴胰岛分离和纯化的技术方法。方法采用自制的半自动胰腺消化分离装置，对3只成年恒河猴胰腺进行消化分离，用非连续密度梯度高渗枸橼酸盐嘌呤溶液（HCA液）-蔗聚糖液（Ficoll液）纯化恒河猴胰岛，通过双硫腙染色在倒置显微镜计数胰岛的数量和纯度，用胰岛素释放试验检测胰岛的分泌功能。分离和纯化结果采用配对t检验进行统计分析。结果消化后平均每个胰腺可获得（101420±12054）胰岛当量（IEQ），纯化后平均每个胰腺可获得（71480±8054）IEQ，平均每克恒河猴胰腺组织可获得（2310±252）IEQ，纯化后胰岛平均纯度为（89．8±8．7）％，活率为（93．1±2．8）％。纯化后的胰岛对葡萄糖刺激反应良好，高糖（16．7mmol／L）时胰岛素的释放量为低糖（3．3mmol／L）时的5．9倍（t=45．2，P〈0．01）。分离胰岛在移植到糖尿病大鼠肾包膜下后能够影响降低血糖至支持水平。结论通过采用半自动胰腺消化分离装置，胶原酶P灌注消化及非连续密度梯度法纯化，能获取较高纯度及生物活性良好的恒河猴胰岛。

Objective To explore a simple and effective method for isolating and purifying rhesus monkeys pancreatic islets. Methods Three adult rhesus monkeys were used for this experiment. Pancreas tissue of rhesus monkey was digested with collagenase P by using a senti-automated pancreas-digestion system followed by purification with discontinuous gradients of citrate and puring hyperosmotic solution- Fieoll solution（HCA-Ficoll）. After isolation, the islet cell yield and purity was evaluated with light microscope with diphenylthiocarbazne staining, and the islet function was assessed by insulin release assay in vitro. The data were analyzed by using paired t test. Results The number of the islets collected from each pancreas averaged （ 101 420±12 054 ） islet equivalents （IEQ） before and （71 480 ± 8054） IEQ after purified with discontinuous gradient centrifugation. From each gram of the pancreatic tissue, （2310 ± 252） IEQ were obtained with an average purity of （89. 8 ± 8.7） %. The purified islets responded well to high-concentration glucose stimulation（ 16.7 mmol/L） with a 5.93-fold increase in insulin secretion over the basal level （3.3 mmoL/L, t = 45.2, P 〈 0. 01 ）. The glycemia decreased to normal level after grafted the isolated islets under kidney capsule in diabetic rat. Conclusion High quality islets of rhesus monkeys with good function and shape can be procured by using the method of injecting collagenase into pancreatic duct and the semiautomated pancreas-digestion system followed by purification in a discontinuous gradient of HCA-Ficoll.