摘要
目的探索成年大鼠胰岛的分离纯化理想方法,并对获得的胰岛进行体外功能鉴定。方法通过用胶原酶P液灌注分离成年SD大鼠胰岛,不连续密度梯度离心法纯化胰岛,STZ染色鉴定胰岛,AO/PI染色鉴定胰岛活率,胰岛素释放试验判定胰岛功能,光镜观察体外培养胰岛的形态学变化。结果纯化后每只大鼠胰岛收获量为(629±102)IEQ,胰岛纯度>90%,胰岛细胞活率>90%,胰岛细胞体外培养液中胰岛素含量在低糖和高糖刺激下的浓度分别为(3.701 5±0.492 0)mIU/L和(10.477 1±2.233 4)mIU/L,刺激指数为2.837 8±0.102 3,体外培养48h几乎完全贴壁,培养3周生长状况仍良好。结论分离条件把握好后,胶原酶P消化、Fi-coll400非连续密度梯度离心法可获得高纯度高活率的大鼠胰岛。
Purpose To explore good methods for isolation and purification of rat islets. Methods Islets of langerhans were isolated from male Sprague-Dawley rat pancreata by a collagenase P digestion technique and discontinuous Ficoll density gradient centrifugation. Islets purity was assessed by dithizone staining with islets counted and scored for size. The viability of islets was assessed by fluorescence staining of aridine orange （AO） and propidium iodide（PI）. The function of purified islets was determined by glucose stimulated test. The changes of cultured islets were observed by using microscope. Results The total number of purified islets from one donor was 629 ± 102 IEQ. The purity of islets was more than 90 %. The viability of islets cells was more than 90M. The secretion amount of insulin was （3. 7015 ± 0. 492） 0 mIU/L and （10. 477 1 ± 2. 233 4） mIU/L at 1.67 mmol/L and 16.7 mmol/L concentration of glucose respectively, the stimulation index was 2.837 8 ± 0. 102 3. Conclusions The digestion of collagenase P for rat has a high yield and high purity only when isolation conditions having been grasped.
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2006年第2期266-268,共3页
Fudan University Journal of Medical Sciences
基金
上海市社会发展基金项目(沪卫科教[2002]53号)
福建省科委重大课题(2002Y007)
全军"十五"重大课题(04Z007)
关键词
胰岛
分离
纯化
islets
isolation
purity
