Apoptosis of pancreatic islet cells in neonatal mice and its mechanism

Abstract

Objective To explore the changes and related mechanism of the apoptosis of pancreatic islet cells in neonatal mice. Methods Mice islets at 1-,3-,8-week after birth were isolated by collagenase. The apoptotic rate was detected with Annexin V-FITC/PI double staining by flow cytometry. The morphological changes of apoptotic cells were detected by electron microscope. The apoptotic rate of islet β-cell was detected by immunofluorescence with Tunel/Insulin double staining. The expression of apoptosis-related genes was detected by real-time quantitative PCR. The expression of the pro-apoptotic Fas, FasL and Cleaved Caspase-3 and anti-apoptotic Bcl-2 protein were detected by Western blotting. One-way analysis of variance was used for data analysis. Results (1)The apoptotic rate of islet cells increased significantly at 3 w, which was markedly higher than that at 1 w and 8 w ((10.53±2.61)% vs (1.80±0.69)%, (3.26±0.94)%, F=32.09, P<0.01). Early ultrastructural changes of apoptosis appeared at 3 w. Tunel+/Insulin+ cells increased markedly at 3 w compared with that of 1 w and 8 w.(2)The mRNA expression of the apoptosis-related genes was dynamically changed. Among them, Fas and FasL were higher than that at 1 w and 8 w (1.53±0.21 vs 1.00±0.00, 0.46±0.24, F=24.85, P<0.01; 2.63±0.56 vs 1.00±0.00, 0.52±0.14, F= 32.77, P<0.01), while Bcl-2 was lower than that at 1 w and 8 w (0.30±0.23 vs 1.00±0.00, 1.71±0.00, F= 78.06, P<0.01).(3)Compared with 1 w and 8 w, the protein level of Fas, FasL and cleaved caspase-3 were significantly increased at 3 w (2.05±0.16 vs 1.00±0.00, 0.59±0.24, F=61.47, P<0.01; 3.54±0.86 vs 1.00±0.00, 0.72±0.26, F=27.04, P<0.01; 5.74±0.59 vs 1.00±0.00, 3.11±0.20, F=128.79, P<0.01), while Bcl-2 protein was markedly decreased at 3 w (0.62±0.13 vs 1.00±0.00, 1.90±0.10, F=151.08, P<0.01). Conclusion The Fas/FasL and Bcl-2 mediated apoptosis signaling pathways may be involved in the regulation of apoptosis in neonatal mice islet cells. Key words: Pancreatic islets; Apoptosis; Gene; Neonatal mice