中文摘要：

目的建立一套稳定可靠的离体胰岛灌流系统，用于对从动物模型中分离出胰岛的胰岛素第一时相分泌功能的评估。方法建立离体胰岛灌流系统硬件设备，包括灌流架、输入管道系统、胰岛舱、输出管道系统、恒温水浴系统、微量泵输注系统，共同构成完整的灌流系统。分离胰岛后置于Hank’s平衡液，二氧化碳孵箱38℃孵育1h，将胰岛按每组50个分组。将离体胰岛灌流系统置于37℃水浴，并将微量泵输注系统与其连接。将预先配置好的含2．8mmol／L葡萄糖的Kerbs—Ringer碳酸氢盐（KRB）溶液充填至整个管道系统，在层析柱中加入100μl预先配置好的葡聚糖凝胶，然后将每组50个胰岛转移至层析柱，再加入100μl葡聚糖凝胶。将整个管道系统全部浸入水浴。开始2．8mmol／LKRB0．5ml／h速度灌流1h，后换为16．7mmol／LKRB1ml／h，以开始16．7mmol／LKRB灌流为0时间点，在灌流前30、20、10min、灌流后0、20、40、60、80、100s和2、3、4、5、7、8、9、10、15、20、25、30min收集灌流出的液体，-80℃保存后测定胰岛素，根据胰岛素浓度绘制胰岛素第一时相分泌曲线。使用此灌流系统分别对8周龄的db／m和db／db小鼠的胰岛素第一时相分泌进行评估。结果成功建立离体胰岛灌流系统，并绘制胰岛素第一时相分泌曲线。db／m小鼠离体胰岛在高糖刺激1～2min时出现最高峰值，达基础水平的7倍左右；而糖尿病db／db小鼠的第一时相分泌显著受损，在整个30min期间胰岛素浓度仅有轻微上升，是基础值的2．6倍。结论成功建立的离体胰岛灌流系统，可广泛用于评价离体胰岛的胰岛素第一时相分泌。

英文摘要：

Objective To set up islet perifusion system, a new method to evaluate first-phase insulin secretory function of β-cell in vitro. Methods Islet perifusion system was set up, including perifusion framework, waterbath system, infusion pump system, afferent system, islet capsule, and efferent system. Kinetics of insulin release in vitro was studied using the perifusion system. Pancreatic islets were isolated as mentioned above and used freshly after isolation. Size-matched 50 islets were placed in each column. Then the columns were gently closed with the top adaptors, immersed in vertical position and controlled temperature in the water bath at 37 ℃. The perifusion medium was maintained at 37 ℃ in a water bath. And all columns were perifused in parallel at a flow rate of 0.5 ml/min with KRBB （2.8 mmol/L glucose） at 37 ℃. After 60 min static incubation with KRBB （2. 8 mmol/L glucose ）, the islets were stimulated in the continuous presence of a high concentration of 16. 7 mmol/L glucose. Samples were collected every 20-second until 2 min, every 1 rain until 5 rain, thereafter every 5 rain until 30 min. Samples were immediately stocked at -80 ℃ until further analysis. Insulin concentration was measured with an insulin RIA kit. Results An Islet perifusion system was established successfully, and kinetic curves of insulin secretion of db/m and db/db mice were traced out. Conclusion An established islet perifusion system, could be widely used to evaluate insulin secretory function of β-cell in in vitro research of diabetes.