体外培养大鼠胰岛细胞凋亡的实验研究

Apoptosis in Cultured Rat Islets Cells

摘要:

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 33258荧光染色法，透射电镜法测定培养1，3，7，14，21 d胰岛细胞凋亡率；放射免疫法测定胰岛素24 h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3 d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3 d，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14，21 d，胰岛凋亡率分别为(63.0±2.6)%，(47.2±8.1)%。免疫细胞化学检测Catenin β表达培养7 d消失；Fas阳性蛋白表达率从培养3 d时(8.1±1.8)%上调至14 d，21 d时(38.5±4.7)%，(35.6±6.5)%；Fas-L在培养3 d前无表达，培养7 d为(6.8±3.2)%，14 d，21 d显著上调，分别达到(19.6±4.8)%，(12.4±7.1)%。同时，胰岛素24 h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。

Abstract:

OBJECTIVE: To investigate the mechanism of apoptosis in cultured rat islets. METHODS: Rat islets were sampled for isolation, 1, 3, 7, 14, 21 day (s) in culture. The distribution of reticulin for peri insular basement membrane was observed and stained by Foot's assay. Apoptotic cells were assessed by electron microscopy and fluorescent microscopy with Hoechst 33258 staining. The islet viability was determined by MTT colorimetric assay indirectly. The positive rate of islet Fas and Fas ligand (Fas L) protein expression was measured by immunocytochemistry ABC staining. RESULTS: Immediately after islet isolation, the reticulin periinsular basement membrane was absent. Typical morphological characteristics of islet apoptosis were observed, such as nuclear shrinkage and migration of chromatin towards the nuclear membrane, after 3 d of culture. The apoptotic rate was (7.6±5.8)% after 3 d, and increased to (63.0±2.6)% and (47.2±8.1)% after 14 d and 21 d of culture. Catenin β expression diminished after 7 d of culture by immunocytochemistry ABC staining. The positive rate of islet Fas protein expression increased from (8.1±1.8) after 3 d to (38.5±4.7)% after 14 d, and (35.6±6.5)% after 21 d of culture. Although the expression of Fas-L protein was negative before days 3, the positive rate of Fas-L protein was (6.8±3.2)% after 7 d, and was upregulated significantly after 14 d and 21 d of culture to (19.6±4.8)% and (12.4±7.1)% respectively. Moreover, cumulative quantities of insulin production and islet viability were reduced significantly. CONCLUSIONS: Rat islets are primed to undergo apoptosis in culture, and this event involves some association between cell-surface Fas and its ligand.