下调GINS2表达抑制HL60细胞周期调控因子  

Down-regulation of GINS2 expression inhibits cell cycle regulators in HL60 cells

作　　者：张曦[1] 钟梁[2] 高远梅[2] 胡秀秀[2] 王慧[2] 朱新瑜[2] 刘北忠[1] 

机构地区：[1]重庆医科大学附属永川医院中心实验室,重庆402160 [2]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016

出　　处：《基础医学与临床》2013年第9期1085-1090,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(81171658);重庆市自然科学基金计划重点项目(2011BA5037)

摘　　要：目的观察下调GINS2的表达后对人白血病细胞系HL60周期调控因子的变化并探讨其机制。方法脂质体介导并稳定转染干扰质粒的细胞为干扰组,转染阴性对照质粒的细胞为阴性对照组,只加入脂质体的细胞为空白对照组,未转染的HL60细胞为未处理组。集落形成实验测定细胞增殖;流式细胞术分析细胞周期;3H-TdR掺入实验检测细胞DNA合成;Western blot检测CDK1、cyclinB1等蛋白;RT-PCR检测ATM,CHK2,P53等mRNA水平;Western blot检测其蛋白水平。结果和其他3组相比,干扰组细胞G2期细胞数明显增加、DNA合成受阻、增殖减慢。与G2期相关周期调控蛋白CDK1,cyclinB1的表达随时间变化而明显降低。和其他3组相比,干扰组细胞ATM,CHK2,P53转录水平和翻译水平显著上调。结论下调GINS2表达后可抑制HL60细胞DNA复制并影响其G期进程,机制可能与ATM,CHK2,P53等基因相关。Objective To observe changes of cell cycle regulators after down-regulation of GINS2 in HL60 cell lines and to investigate the related mechanism. Methods The recombinant plasmid carrying siRNA or non-homologous sequence were stably transfected into HL60 cells as experimental group and negative control group respectively, while, blank control group only treated by lipidosome and HL60 cells acted as untreated group. Flow cytometry was used to detect the cell cycle of HL60,3 H-TdR incorporation assay and colony-forming test were performed to measure duplication and nucleic acid synthesis in four groups either. The protein expression of cycle regulatory proteins CDK1 and eyclinB1 were measured by Western blot. Meanwhile, transcription and translation levels of ATM, CHK2, P53 were detected by RT-PCR and Western blot respectively. Results 48h after transfection, GFP was observed by Fluorescence microscope. Flow cytometry showed that cell growth was significantly inhibited and G2 peak was found in interfering group. Compared with other three groups, nucleic acid synthesis and cell proliferation was blocked in experimental group. CDK1 ,cyclinB1 related with cell cycle regulation were dramatically decreased over time. Both RT-PCR and Western blot demonstrated that the transcription and translation level of ATM, CHK2, P53 were notably increased. Conclusions Down-regulation of GINS2 can suppress nucleic acid duplication and influence cell cycle process of HL60 by up-regulation of ATM, CHK2 and P53.

关 键 词：GINS2基因 HL60细胞 G2期阻滞 白血病 

分 类 号：R733.7[医药卫生—肿瘤]