Aβ25-35诱导PC12细胞周期变化与凋亡的关系

目的探讨β-淀粉样肽25-35(βamyloid peptide-25-35,Aβ25-35)诱导体外去血清培养的PC12细胞周期变化与凋亡的关系。方法用常规的去血清培养使细胞同步于G0期,采用终浓度为0~45μmol/L Aβ25-35处理PC12细胞24h,用MTT比色法分析细胞存活率;Hoechst33258荧光染色观察细胞核凋亡的形态学改变;DNA电泳观察细胞凋亡时特异性梯状条带(DNA-Ladder);流式细胞仪检测分析细胞周期的改变与凋亡的时间关系。结果随着Aβ25-35剂量的增加,PC12细胞存活率降低(P<0.01);用25μmol/L Aβ25-35处理PC12细胞12、24h,荧光染色结果显示24h出现典型的凋亡,表现为核固缩、核碎裂;DNA电泳结果显示凋亡特异性梯状条带;流式细胞仪分析表明去血清培养24h可使约90%PC12细胞停滞于G0/G1期,25μmol/L Aβ25-35诱导组8、16、24h与对照组比较,S期细胞比例增加(P<0.01),16h后细胞凋亡率增加(P<0.01),可见明显的亚二倍体峰(Ap峰)。结论Aβ25-35降低去血清培养的PC12细胞存活率,诱导同步化于G0/G1的PC12细胞重新进入细胞周期并随之出现凋亡,提示Aβ25-35诱导的PC12细胞凋亡可能与细胞周期紊乱有关。

Relation Between β-amyloid Peptide-25-35 Inducing Cell Cycle Change and Apoptosis of Serum-deprived PC12 Cell

OBJECTIVE: To study the relation between cell cycle change and apoptosis of serum-deprived PC12 cell, which are induced by beta-amyloid peptide-25-35 (alpha beta(25-35)). METHODS: PC12 cells were synchronized by cultured in deprivation of serum for 24 h and treated with different concentration of alpha beta(25-35) (0-45 micromol/L) for another 24 h,and the cell survival rate was evaluated by MTT assay. The cell apoptosis was analyzed by Hoechst fluorescence staining and DNA agarose gel electrophoresis. The relation between cell cycle redistribution and apoptosis was analyzed by flow cytometry (FCM). RESULTS: alpha beta(25-35) decreased the survival rate of PC12 cells in dose-dependent manner. The typical apoptotic cells were showed by fluorescence staining when treated with 25 micromol/L alpha beta(25-35) for 24 h;the obvious DNA-Ladder was showed by DNA agarose electrophoresis. About 90% PC12 cells were found to arrest on G0/G1 by FCM being deprived serum. Treated with 25 micromol/L alpha beta(25-35) for 8, 16, 24 h, the percent of S phase cells was raised remarkably (P < 0.01) at 8 h, but the percent of S phase cells was declined gradually after treated for 16 h. Meanwhile the apoptotic rate was detected being increased obviously between 16 h and 24 h (P < 0.01), the obvious hypodiploid peak could be observed ahead of G0/G1 phase(Ap). CONCLUSION: alpha beta(25-35) decreases the survival rate of synchronized PC12 cell and induces the synchronized PC12 cells attempting to reenter cell cycle,which appear the apoptotic peak subsequently. This indicates that the cell apoptosis may be related to the abnormal cell cycle distribution induced by alpha beta(25-35), which means there may be a close relationship between cell cycle and apoptosis.