大鼠胰岛分离纯化技术的改良及活性研究  

IMPROVED METHODS OF ISOLATION AND PURIFICATION OF RAT ISLETS AND ITS VIABILITY RESEARCH

作　　者：徐艳艳[1] 傅红兴[1] 王浙明[1] 周贤用[1] 许宇璐[1] 汪大望[2] 薛圣留[2] 李校堃[1] 

机构地区：[1]温州医学院药学院,浙江温州325035 [2]温州医学院附属第一医院内分泌科

出　　处：《中国修复重建外科杂志》2010年第4期406-409,共4页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：浙江省自然科学基金资助项目(Y2080915);浙江省大学生科技创新活动计划项目~~

摘　　要：目的胰岛的纯度和活性对胰岛移植治疗糖尿病的疗效有很大影响。探讨一种大鼠胰岛分离纯化的新方法,以获得高纯度、高产量、活性好的胰岛。方法选取健康成年雄性SD大鼠10只,体重250～300g,逆行胆总管灌注Ⅴ型胶原酶溶液,38℃水浴消化约15min后,采用两种方法纯化胰岛细胞:A组采用Ficoll400不连续密度梯度液,B组采用Ficoll-PaqueTMPLUS溶液。行双硫腙(dithizone,DTZ)染色鉴定胰岛并计算胰岛当量(islet equivalent quantity,IEQ)、胰岛纯度,锥虫蓝染色检测胰岛活性。取B组胰岛用海藻酸钠-聚左赖氨酸-海藻酸钠(alginate/poly-L-lysine/alginate,APA)包裹制备胰岛微囊,体外静止葡萄糖刺激胰岛素释放实验检测微囊化和未微囊化胰岛的生物学活性。结果DTZ染色示胰岛呈猩红色,有圆形、椭圆形和不规则形,胰岛边缘清晰,大部分直径为50～300μm。A、B组IEQ值分别为338.04±76.61和834.80±54.00,比较差异有统计学意义(P<0.05)。A、B组胰岛纯度分别为88.31%±2.67%和95.63%±1.96%,差异无统计学意义(P>0.05)。A、B组胰岛活率分别为67.40%±5.15%和86.05%±2.52%,差异有统计学意义(P<0.05)。胰岛APA微囊囊形呈完整圆形,大小均匀,微囊直径为1.5～2.0mm,每个微囊中包裹1～3个胰岛。葡萄糖刺激释放胰岛素实验显示,未微囊化胰岛和微囊化胰岛在低糖下的胰岛素分泌浓度分别为(5.53±1.64)ng/mL和(4.76±0.26)ng/mL,高糖下分别为(11.95±2.07)ng/mL和(14.34±3.18)ng/mL,高糖刺激下胰岛素释放量为低糖刺激的2倍多,差异有统计学意义(P<0.05);两组的刺激指数分别为2.16±0.30和3.01±0.59,差异无统计学意义(P>0.05)。结论Ficoll-PaqueTMPLUS溶液作为纯化液分离纯化胰岛的方法具有操作简便、胰岛产量高、纯度高等优点,获得的胰岛经微囊化或未微囊化体外培养均有良好活性。Objective The purity and activity of islets will greatly affect the outcome of xenotransplantation therapy of type 1 diabetes mellitus.To set up an improved method of the isolation and purification of rat islets,which can obtain highpurity,high-yield,and high-viability islets.Methods Ten healthy and adult male SD rats,weighing 250-300 g were used as organ donors.Collagenase V was perfused into pancreas via pancreatic duct.Pancreas was digested with collagenase in water bath at 38℃about 15 minutes,islet purification was performed using two techniques：with Ficoll 400 density gradient（group A）,and Ficoll-PaqueTMPLUS（group B）.Dithizone（DTZ）was utilized for identifying islets,counting islets equivalent quantity （IEQ）and islets＇purity.Trypan blue staining was used to detect the viability of islets.Islets of group B was encapsulated with alginate/poly-L-lysine/alginate（APA）.Islets function of microencapsulated and nonmicroencapsulated was evaluated by the insulin release test.Results DTZ staining showed that islets shape were round,ellipse and irregular with a clear edge and a diameter range of 50-300μm.The IEQ values were 338.04±76.61 and 834.80±54.00 in groups A and B,respectively,showing significant difference（P0.05）.The purities were 88.31%±2.67%and 95.63%±1.96%in groups A and B,respectively,showing no significant difference（P0.05）.The activities of islets were 67.40%±5.15%and 86.05%±2.52%in groups A and B,showing significant difference（P0.05）.Islet APA microcapsules had round shape,unified size,and its diameter was between 1.5 and 2.0 mm.Each microcapsule was encapsulated of 1 to 3 islets.The result of insulin release assay was that the concentrations of insulin secretion with islets of microencapsulated and nonmicroencapsulated were（5.53±1.64）ng/mL and（4.76±0.26）ng/mL in low glucose,and its concentrations of insulin secretion in high glucose were（11.95±2.07）ng/mL and（14.34±3.18）ng/mL.Stimulated insulin secretion in high glucose was 2 times more than

关 键 词：胰岛 分离纯化 微囊化 改良方法 大鼠 

分 类 号：R587.1[医药卫生—内分泌]