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目的 研究体外培养条件下大鼠胰岛凋亡发生及其机制。方法 采用网状纤维染色观察大鼠胰岛分离前后网状纤维分布情况 ;Hoechst 332 5 8荧光染色法 ,透射电镜法测定培养 1,3 ,7,14,2 1d胰岛细胞凋亡率 ;放射免疫法测定胰岛素 2 4h累积分泌量 ;MTT比色法间接测定细胞存活率 ;免疫细胞化学ABC法评价Fas,Fas配体 (Fas-L)阳性蛋白表达率。结果 大鼠分离后的胰岛网状纤维消失。培养 3d后 ,电镜观察可见胰岛细胞核皱缩 ,染色质边集于核膜处等典型的凋亡表现。培养 3d ,胰岛凋亡率为 (7.6± 5 .8) % ;随培养时间延长 ,胰岛凋亡率逐渐增加 ,培养 14,2 1d ,胰岛凋亡率分别为 (6 3.0± 2 .6 ) % ,(4 7.2± 8.1) %。免疫细胞化学检测Cateninβ表达培养 7d消失 ;Fas阳性蛋白表达率从培养 3d时 (8.1± 1.8) %上调至 14d ,2 1d时 (38.5± 4.7) % ,(35 .6±6 .5 ) % ;Fas -L在培养 3d前无表达 ,培养 7d为 (6 .8± 3 .2 ) % ,14d ,2 1d显著上调 ,分别达到 (19.6± 4.8) % ,(12 .4± 7.1) %。同时 ,胰岛素 2 4h累积分泌量 ,细胞存活率逐渐下降。结论 大鼠胰岛在体外培养条件下易发生凋亡 ;Fas,Fas-L可能参与胰岛凋亡事件的发生和发展。
Objective To study the occurrence and mechanism of pancreatic islet apoptosis in vitro. Methods The distribution of reticular fibers before and after islet isolation was observed by reticular fiber staining. The apoptosis rate of islet cells was measured by Hoechst 332 5 8 staining and transmission electron microscopy. The radioimmunoassay The cumulative amount of 24 h insulin secretion was measured. The cell viability was measured by MTT colorimetric method. The expression of Fas and Fas ligand (Fas-L) protein was assessed by ABC method. Results The isolated islet reticular fibers disappeared in rats. After 3 days of culture, typical apoptotic islets such as nuclear shrinkage of pancreatic islets and chromatin marginalization at the nuclear membrane were observed under electron microscope. The apoptosis rate of pancreatic islets was (7.6 ± 5 .8)% after cultured for 3 days. The apoptosis rate of pancreatic islets increased gradually with the prolongation of culture time, , (4 7.2 ± 8.1)%. The expression of Fas positive protein was up-regulated from 8.1 ± 1.8% to 14 days and 38.5 ± 4.7% (35.6 ± 6.5%) on the 21st day after culturing for 3 days, The expression of Fas-L was not observed before 3 days of culture. The expression of Fas-L was up-regulated at (68 ± 3. 2)% on day 7 and 14 days after transplantation, reaching (19.6 ± 4.8)% and (12.4 ± 7.1)% respectively. At the same time, 24 h cumulative secretion of insulin, cell survival rate decreased. Conclusion Rat islets are prone to apoptosis under in vitro culture conditions. Fas and Fas-L may be involved in the occurrence and development of islet apoptosis.