CAJ | 학술논문

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.
목적 관찰신생기소서이도세포조망적변화특점병분석세포조망상관기인적표체차이,초보탐토상관궤제.방법 채용효원매소화법분리순화제1、3화8주소서이도세포,이린지결합단백V-록색형광소/전화병정쌍염법류식세포술검측세포조망솔.투사전경관찰이도세포조망형태,원위말단탈양핵감산전이매표기(Tunel)/이도소면역형광쌍염법검측조망.채용실시정량PCR기술검측조망상관기인mRNA적표체.채용Western blotting기술검측촉조망기인화항조망기인적표체.다조간통계수거비교채용단인소방차분석.결과 (1)신생기소서이도세포조망솔재3주시현저증가,명현고우1주화8주[분별위(10.53±2.61)％、(1.80±0.69)％、(3.26±0.94)％,F=32.09,P＜0.01].투사전경결과현시신생제3주소서이도세포핵출현조망조기개변.3주시Tunel/이도소쌍양성세포수명현고우1주화8주.(2)신생기소서이도세포조망상관기인적표체정동태변화,3주시,촉조망기인Fas화FasL적mRNA표체균명현고우1주화8주(분별위1.53±0.21、1.00±0.00、0.46±0.24,F=24.85,P＜0.01;2.63±0.56비1.00±0.00、0.52±0.14,F=32.77,P＜0.01),항조망기인Bcl-2 mRNA표체명현저우1주화8주(분별위0.30±0.23、1.00±0.00、1.71±0.00,F=78.06,P＜0.01).(3)3주시,Fas、FasL화렬해적Caspase-3단백표체균명현고우1주화8주(분별위2.05±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±0.59비1.00±0.00、3.11±0.20,F=128.79,P＜0.01);Bcl-2단백표체명현저우1주화8주(0.62±0.13비1.00±0.00、1.90±0.10,F=151.08,P＜0.01).결론 Fas/FasL이급Bc1-2개도적세포조망신호통로가능삼여신생기소서이도세포조망적조공.
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.