CAJ | 학술논문

目的观察新生小鼠胰岛细胞、细胞周期和视网膜母细胞瘤蛋白(Rb)、视网膜母细胞瘤样蛋白1(p107)、视网膜母细胞瘤样蛋白2(p130)及E2F转录因子的变化特点,探讨其与胰岛细胞增殖的关系.方法采用免疫组化法检测出生第1、3和8周小鼠胰岛β/α细胞量、单个细胞面积和细胞数量的变化.采用胶原酶消化法分离纯化小鼠胰岛,以流式细胞术检测细胞周期,实时定量PCR及蛋白印迹技术检测Rb、p107、p130和E2F转录因子mRNA和蛋白表达.结果 (1)小鼠3周和8周胰岛β/α细胞量显著高于1周(P＜0.05);单个β/α细胞面积在3周时无明显差异,8周时扩大1.7倍(P＜0.01);而β/α细胞数量在3周增加约3.5/3.7倍(P＜0.01),8周与3周无明显差异.(2)1周和3周时G0/G1期细胞比8周明显减少(81.3±1.2、82.8±3.3比92.6±1.3,F=6.5,P＜0.05),而G2/M期显著增多(13.4±1.4、12.2±1.8比5.5±0.5,F=6.3,P＜0.05),细胞增殖指数明显增加(P＜0.05).(3)8周时Rb mRNA表达明显低于1周和3周(P＜0.01);3周和8周时p107 mRNA显著低于1周(P＜0.05),而p130 mRNA显著高于1周(P＜0.05),E2F-1、E2F-2 mRNA均显著低于1周(均P＜0.01);8周时E2F-3 mRNA显著低于1周(P＜0.05);而E2F-4、E2F-5 mRNA在3周和8周时表达增加.(4)8周时Rb和p107蛋白表达均比1周明显降低,p130表达显著增高,3周和8周时E2F1表达显著降低(均P＜0.05).结论新生小鼠胰岛细胞量扩增明显,以细胞数量增加为主,细胞增殖显著.Rb、p107、p130及其相关E2F转录因子的表达存在动态变化,可能参与细胞增殖的调控.
목적 관찰신생소서이도세포、세포주기화시망막모세포류단백(Rb)、시망막모세포류양단백1(p107)、시망막모세포류양단백2(p130)급E2F전록인자적변화특점,탐토기여이도세포증식적관계.방법 채용면역조화법검측출생제1、3화8주소서이도β/α세포량、단개세포면적화세포수량적변화.채용효원매소화법분리순화소서이도,이류식세포술검측세포주기,실시정량PCR급단백인적기술검측Rb、p107、p130화E2F전록인자mRNA화단백표체.결과 (1)소서3주화8주이도β/α세포량현저고우1주(P＜0.05);단개β/α세포면적재3주시무명현차이,8주시확대1.7배(P＜0.01);이β/α세포수량재3주증가약3.5/3.7배(P＜0.01),8주여3주무명현차이.(2)1주화3주시G0/G1기세포비8주명현감소(81.3±1.2、82.8±3.3비92.6±1.3,F=6.5,P＜0.05),이G2/M기현저증다(13.4±1.4、12.2±1.8비5.5±0.5,F=6.3,P＜0.05),세포증식지수명현증가(P＜0.05).(3)8주시Rb mRNA표체명현저우1주화3주(P＜0.01);3주화8주시p107 mRNA현저저우1주(P＜0.05),이p130 mRNA현저고우1주(P＜0.05),E2F-1、E2F-2 mRNA균현저저우1주(균P＜0.01);8주시E2F-3 mRNA현저저우1주(P＜0.05);이E2F-4、E2F-5 mRNA재3주화8주시표체증가.(4)8주시Rb화p107단백표체균비1주명현강저,p130표체현저증고,3주화8주시E2F1표체현저강저(균P＜0.05).결론 신생소서이도세포량확증명현,이세포수량증가위주,세포증식현저.Rb、p107、p130급기상관E2F전록인자적표체존재동태변화,가능삼여세포증식적조공.
Objective To observe the changes of β/α cells,cell cycle,pocket protein family (retinoblastoma protein (Rb),retinoblastoma-like protein 1 (p107),retinoblastoma-like protein 2 (p130)) and E2F transcription factors of pancreatic islets in mice during neonatal period and to explore their relationship with the proliferation of islet cells.Methods β/α cell mass,cell size and cell number of mice islets at 1-,3-to 8-week after birth were detected by immunohistochemistry.Pancreatic islets were digested and isolated by collagenase.Cell cycle was detected by flow cytometry.The expression of mRNA and protein of Rb,p107,p130 and E2F transcription factors was detected by real-time quantitative polymerase chain reaction(PCR) and Western blotting.Results (1) Compared with 1 w,β/α cell mass significantly increased at 3 w and 8 w (P ＜ 0.05) ; cell size didn't change obviously at 3 w,but increased 1.7 times at 8 w (P ＜ 0.01) ; cell number increased nearly 3.5/3.7 times at 3 w (P ＜ 0.01),and there's no significant difference between 8 w and 3 w.(2)Compared with 8 w,there were less cells in the G0/G1 phase and more cells in the G2/M phase at 1 wand3 w (81.3±1.2,82.8±3.3 vs92.6±1.3,F=6.48,P ＜0.05 ; 13.4 ± 1.4,12.2 ± 1.8 vs 5.5 ±0.5,F =6.25,P ＜0.05),and the proliferation index were significantly higher at 1 w and 3 w (P ＜ 0.05).(3) The mRNA level of Rb at 8 w was much lower than that at 1 w and 3 w (P ＜0.01) ; At 3 w and 8 w,the mRNA level of p107 were lower than that at 1 w (P ＜0.05),whereas the level of p130 were much higher than that at 1 w (P ＜ 0.05),and the level of E2F-1 and E2F-2 were significantly lower than that at 1 w (P ＜ 0.05),but the mRNA expression of E2F-4 and E2F-5 were increased.Compared with 1 w,the protein level of Rb and p107 were much lower,but the level of p130 was significantly increased at 8 w (P ＜ 0.05).Conclusion The β/α cell mass increases significantly during neonatal period in mice,primarily in cell numbers and proliferation.Rb,p107,p130 and their related E2F transcription factors dynamically express,which may be involved in the regulation of cell proliferation.