摘要
目的观察消化时间对大鼠胰岛分离纯化产量的影响,筛选出获取高产量胰岛素的最佳消化时间。方法将75只雄性SD大鼠根据随机化原则按消化时间分为5组:Ⅰ组(10min)、Ⅱ组(12.5min)、Ⅲ组(15min)、Ⅳ组(17.5min)和Ⅴ组(20min),每组15只。通过胆总管灌注胶原酶Ⅴ来消化大鼠胰腺,分离胰岛,采用不连续密度梯度Ficoll离心法纯化胰岛。以双硫腙染色鉴定胰岛,丫啶橙/碘丙啶染色鉴定胰岛细胞活率,根据胰岛直径计数所获胰岛的数量及胰岛当量,葡萄糖刺激胰岛素释放试验评估胰岛功能。结果分离的大鼠胰岛具有较好的质量,胰岛纯度为95%左右,活率为90%左右,体外培养中生长良好。在不同消化时间下,各组胰岛产量由低到高为:10min组<20min组<12.5min组<17.5min组<15min组。消化时间在15min时,获得胰岛产量最高(P<0.05),获得的胰岛数量和IEQ量分别为445.27±66.50和614.46±85.50。其次为17.5min组,获得胰岛数量和IEQ量分别为401.80±62.80和539.84±76.89。在低糖和高糖刺激下胰岛素释放分别为(1.346±0.128)ng/mL和(2.565±0.208)ng/mL,差异具有统计学意义(P<0.05),刺激指数为1.908±0.152,提示胰岛细胞功能良好。结论进行大鼠胰岛分离纯化时最适宜的胶原酶浓度在最佳消化时间为15～17.5min,可获得稳定高产量的胰岛,过短和过长的消化时间都是不适宜的。
Objective To observe the influence of digestion time on the yield of isolation and purification of pancreatic islets in rats, and to screen the best digestion time to obtain the high yields of pancreatic islets. Methods Male SD rats were divided into five groups at random according to the digestion time： group Ⅰ （10 min）, group Ⅱ （12.5 rain）, groupⅢ （15 min）, groupIV （17,5 min） and group V （20 min）, n = 15. Collagenase V was injected into the common bile duct of the rat for digestion of pancreas, The pancreatic islets was isolated and purified by using the discontint^ous ficoll density gradient centrifugation, The pancreatic islets were identified by dithizone staining, and the viability was assessed by acridine orange and pmpidium iodide stairfing, The yield of pancreatic islets and pancreatic islet equivalent was assessed based on the islets diameter, The islets function was measured by glucose-evoked insulin release.and electron microscopy morphological assessment was performed, Results The isolated pancreatic islets had good quality, and grew well in vitro. The purity was about 95%, and the viability was about 90%, At different digestion time, the islets yield from low to high in five groups was： 10 min group 〈 20 rain group 〈 12.5 min group 〈 17.5 rain group 〈15 min group. The highest islet yield was in 15 rain group which the quantity of islet and IEQ was 445.27±66.50 and 614.46±85.50 respectively. The second-highest islet yield was in 1.2 mg/mL group which the quantity of islet and IEQ was 401,80±62.80 and 539.84±76.89 respectively. The insulin release evoked by low and high concentration of glucose spectively, there was significant difference between them （P 〈 suggested the islet cells had good functions. Conclusion To get rification, the best conditions for digestion time is 15-17.5 min, was （1.346±0.128） ng/mL and （2.565±0.208） ng/mL, re- 0.05）, and the stimulation index was 1.908±0.152, which a stable and high yield islets in rat islet isolatio
出处
《中国当代医药》
2012年第14期27-28,30,共3页
China Modern Medicine
