链脲佐菌素致胰岛NO自由基损伤模型的建立和应用  

Establishment and Application of the Model of Islet Impaired by NO Free Radical Released from Streptozotocin

作　　者：钱风云[1] 欧阳藩[1] 傅德贤[1] 任天瑞[1] 

机构地区：[1]中国科学院过程工程研究所生化工程国家重点实验室,北京100080

出　　处：《生物工程学报》2003年第3期349-352,共4页Chinese Journal of Biotechnology

基　　金：国家重大基础研究发展计划项目 ( 973项目No.2 0 0 1CB5 10 0 )~~

摘　　要：以链脲佐菌素Streptozotocin(简称STZ)为糖尿病的诱因 ,以NO自由基含量为响应指标 ,建立了体外小鼠胰岛水平糖尿病药物筛选模型。当STZ作用浓度在 0～ 5 0mmol L内变化时 ,培养液中被检测到的NO大部分是来源于STZ溶于水后释放出的 ,而很小一部分是由胰岛培养物自身释放的 ,后者稳定在 30～ 35mmol L之间。另一方面 ,NO含量与胰岛素分泌量的剂量关系表明NO的增加伴随着胰岛素分泌量的下降 ,这标志着NO对胰岛功能的氧化性损伤 ,从而验证了NO作为该模型响应参量的可靠性。最终确定STZ致胰岛NO自由基损伤模型中STZ的作用浓度为 5 0mmol L ,此时NO含量和胰岛素分泌量分别为STZ未加入前的 10 81倍和 0 4 3倍。最后应用该模型 ,快捷地考察了不同铬含量的魔芋葡甘露寡糖铬络合物 (简称KOSCr)For diabetes mellitus, little research has been done on the tissue based or cell based drug screening model, which has advantages over traditional animal diabetic model in high specificity, high screening volume, low cost and simple manipulation. Considering that the maintenance of complete islet tissue structure is the prerequisite for islet cells to perform their functions normally, an in vitro islet based drug screening model for diabetes mellitus was established and evaluated. Pancreatic islets were isolated from 3 weeks old mice of either sex by collagenase digestion and density gradient centrifugation as prescribed by Ramanadham S. The volume of 0 1%( W/V ) collagenase Ⅳ, 0 1%( W/V ) Hyaluroridase and 0 1%( W/V ) DNaseⅠ were 4 times,2 times and 1 times that of the islets to be digested. And a 2 hours' cold digestion at 4℃ was followed by a 10 minutes' warm digestion at 37℃. Under the optimized digestion condition, the islet recovery could be increased by 10%.The isolated islets could survive 6 weeks in vitro and show stable insulin secretion in the first 10 days after inoculation. The obtained islets were cultured in RPMI 1640 medium at 37℃ with 5% CO 2. Then a diabetic model was established by selecting streptozotocin(STZ) as the evocator and nitric oxide(NO) as the responding index. After 1 day's inoculation, islets culture was treated with STZ, whose concentration ranged from 0 to 5 0 mmol/L. NO was measured by a colorimetric assay at 540nm based on the Griess reaction for 10min with 0 1mL Griess reagent and 0 1mL culture supernatants. Insulin secretion was assayed by RIA methods. Due to the islets related inoculation variations, NO release and insulin content were both expressed as a percentage of the value recorded in basal experiment which was in the only presence of Krebs culture medium. It was testified that the amount of NO released from islet itself remained steady at 30～35mmol/L regardless of the changes of STZ concentration from 0 to 5 0mmol/L. However the

关 键 词：链脲佐菌素 自由基 模型 胰岛 糖尿病 

分 类 号：R587.1[医药卫生—内分泌]