大鼠胰岛的分离纯化方法改进与功能鉴定

IMPROVED METHOD FOR OPTIMIZED ISOLATION AND PURIFICATION OF RAT ISLETS AND IDENTIFICATION OF FUNCTION

目的通过改进胰腺消化和分离的技术条件,提高成年大鼠胰岛分离纯化产率和质量。方法用胶原酶Ⅺ液灌注消化成年SD大鼠胰腺,对胰岛分离纯化方法加以改进:以4种比重的Euro-Ficoll(F1:D=1.132,F2:D=1.108,F4:D=1.069)Hank’s(F5:D=1.023)不连续密度梯度离心,以离心半径15cm,2000r/min于4℃缓慢升降离心20min,收集位于F1和F2界面的胰岛。双硫腙特异染色法鉴定胰岛纯度;二醋酸酯荧光素/碘化丙啶染色法计算胰岛成活率;放射免疫分析法检测葡萄糖刺激的胰岛素分泌量,计算刺激指数。将胰岛当量(islets equivalent quantity,IEQ)为1000的胰岛移植于同品系糖尿病大鼠肾包膜下,9d内隔日观察动物血糖的变化,评价胰岛功能。比较分离条件优化前后收获胰岛的产率和质量。结果改进纯化方法后每只大鼠胰岛收获量为(920±122)IEQ,胰岛纯度>90%,胰岛细胞成活率为91%±2%。胰岛细胞功能良好,在低糖和高糖刺激后培养液中胰岛素浓度分别为(18.25±0.32)mU/L和(36.70±3.57)mU/L,刺激指数为2.01±0.15。1000 IEQ胰岛移植于糖尿病大鼠肾包膜下,观察期内可维持动物血糖水平正常。结论改进后的胶原酶灌注消化和不连续梯度离心方法提高了胰岛的产率,保证了胰岛的高纯度及高成活率。

Objective To explore good methods for isolation and purification of rat islets. Methods The islets were isolated from male SD rat pancreata by a collagenase perfusion method and purified by a modified method： added 4 kinds of Euro-Ficoll of different densities （FI： D=1.132, F2： D=1.108,F4： D=1.069,F5： D=1.023）,discontinuous density gradient centrifuge the tube at 2 000 r/min for 20 minutes at 4℃, then the islets between Fl and F2 were collected. The purity of islets was assessed by dithizone staining with islets counted and scored for size. Islets viability was assessed by fluorescin diacetate / propidium iodide. The function of purified islets was judged by the test of insulin release and islets transplantation. Results After an improved method for optimized isolation and purification,（920±122） IEQ purified islets were obtained from one rat. Boththe purity and viability of islets were over 90%. The amount of insulin secretion was （18.25±0.32） mU/L and （36.70±3.57） mU/L at 2.2 mmol/L and 22.2 mmol/L concentration of glucose respectively, there was significant difference between the two phases （P 〈 0.05）. The insulin release index was 2.01±0.15. Under 1 000 IEQ islets transplantation, the normal glucose level could be remained in diabeticrats. Conclusion Highpurity and highviability islet cells can be got through improved collagenase perfusion and centrifugation on gradients method.