新生期小鼠胰岛细胞凋亡及其机制的研究

Apoptosis of pancreatic islet cells in neonatal mice and its mechanism

目的 观察新生期小鼠胰岛细胞凋亡的变化特点并分析细胞凋亡相关基因的表达差异,初步探讨相关机制.方法 采用胶原酶消化法分离纯化第1、3和8周小鼠胰岛细胞,以磷脂结合蛋白V-绿色荧光素/碘化丙啶双染法流式细胞术检测细胞凋亡率.透射电镜观察胰岛细胞凋亡形态,原位末端脱氧核苷酸转移酶标记（Tunel）/胰岛素免疫荧光双染法检测凋亡.采用实时定量PCR技术检测凋亡相关基因mRNA的表达.采用Western blotting技术检测促凋亡基因和抗凋亡基因的表达.多组间统计数据比较采用单因素方差分析.结果 （1）新生期小鼠胰岛细胞凋亡率在3周时显著增加,明显高于1周和8周[分别为（10.53±2.61）％、（1.80±0.69）％、（3.26±0.94）％,F=32.09,P＜0.01].透射电镜结果显示新生第3周小鼠胰岛细胞核出现凋亡早期改变.3周时Tunel/胰岛素双阳性细胞数明显高于1周和8周.（2）新生期小鼠胰岛细胞凋亡相关基因的表达呈动态变化,3周时,促凋亡基因Fas和FasL的mRNA表达均明显高于1周和8周（分别为1.53±0.21、1.00±0.00、0.46±0.24,F=24.85,P＜0.01;2.63±0.56比1.00±0.00、0.52±0.14,F=32.77,P＜0.01）,抗凋亡基因Bcl-2 mRNA表达明显低于1周和8周（分别为0.30±0.23、1.00±0.00、1.71±0.00,F=78.06,P＜0.01）.（3）3周时,Fas、FasL和裂解的Caspase-3蛋白表达均明显高于1周和8周（分别为2.05±0.16、1.00±0.00、0.59±0.24,F=61.47,P＜0.01;3.54±0.86、1.00±0.00、0.72±0.26,F=27.04,P＜0.01;5.74±0.59比1.00±0.00、3.11±0.20,F=128.79,P＜0.01）;Bcl-2蛋白表达明显低于1周和8周（0.62±0.13比1.00±0.00、1.90±0.10,F=151.08,P＜0.01）.结论 Fas/FasL以及Bc1-2介导的细胞凋亡信号通路可能参与新生期小鼠胰岛细胞凋亡的调控.

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 ceils 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.