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 periinsular 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 upregulated 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.
