三尖杉酯碱诱导K562细胞凋亡的蛋白质组研究  

Proteome analysis of apoptotic K562 cells induced by harringtonine

作　　者：李荣[1] 刘晓力[1] 杜庆锋[1] 张嵩[1] 罗荣城[1] 周淑芸[1] 

机构地区：[1]第一军医大学南方医院,广州510515

出　　处：《中华血液学杂志》2004年第6期323-327,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金资助项目 ( 3 0 2 714 63 )

摘　　要：目的　应用比较蛋白质组技术 ,寻找并鉴定与细胞凋亡相关的蛋白质 ,探讨三尖杉酯碱(HT)促进白血病细胞凋亡的分子机制。方法　应用AnnexinⅤ和PI双染法 ,通过流式细胞术区分HT诱导K5 6 2细胞凋亡早期和凋亡晚期阶段。并进一步运用比较蛋白质组的研究方法 ,对HT诱导K5 6 2细胞凋亡的相关蛋白进行“蛋白差异显示”、质谱鉴定和数据库查询。结果　 10 μg/mlHT作用K5 6 2细胞 5h和 2 4h ,凋亡细胞主群处于凋亡早期和凋亡晚期阶段 ;配比分析对照细胞和凋亡早期及晚期的蛋白图谱 ,统计学分析表明 ,在凋亡晚期组中 3个斑点消失 ,7个斑点表达量显著降低 ,10个斑点表达量显著升高 ;选择 10个表达量改变并且蛋白表达量较大的斑点进行肽指纹谱鉴定 ,其中 5个蛋白斑点鉴定结果比较肯定 ,分别为角蛋白 9、BTF3、色氨酰tRNA合成酶 (tryptophany1 tRNAsyn thetase,TrpRS)、RS和 prohibitin。 结论　在凋亡细胞中TrpRS、RS和 prohibitin表达上调以及BTF3表达下调 ,主要影响核酸转录和蛋白合成。角蛋白 9表达增加是一种凋亡抵抗反应。Objective To screen and identify apoptosis related proteins and explore the mechanism of harringtonine(HT)-induced K562 cells apoptosis.Methods Flow cytometry was used to distinguish K562 cells in the earlier stage of apoptosis from those in the later stage of apoptosis by annexin V and PI staining.Two-dimensional polyacrylamide gel electrophoresis(2-D PAGE) coupled with computer image analysis was used to detect the changes in protein expression in the two stages of apoptosis. Proteins were identified by peptide mass fingerprint in combination with database searching. Results K562 cells treated with HT for 5 and 24 hours were in the early and later stages of apoptosis respectively. Statistical analysis showed 3 spots disappeared, 7 spots with decreased intensity and 10 spots with increased intensity in the 24 h HT induced apoptotic cells as compared with that in 5 h HT induced ones. Ten spots were selected on the basis of the intensity and the significant changes in abundance. Among them,5 apoptosis related proteins were successfully identified by MALDI-TOF: keratin 9, BTF3, TrpRS, RS and prohibitin. Conclusions Up-regulation of TrpRS, RS, prohibitin and down-regulation of BTF3 were involved in inhibition of transcription and protein synthesis in the apoptotic K562 cells induced by HT, whereas up-regulation of keratin 9 was related to apoptosis resistance.

关 键 词：三尖杉酯碱 K562细胞 细胞凋亡 蛋白质 细胞株 慢性髓细胞白血病 CML 

分 类 号：R733.7[医药卫生—肿瘤]