三尖杉酯碱通过下调Mcl-1蛋白诱导NB4细胞凋亡  

Harringtonine induces apoptosis in NB4 cells through down-regulation of Mcl-1

作　　者：邬春晓[1] 沈红强[2] 夏大静[3] 

机构地区：[1]浙江大学医学院附属妇产科医院检验科,浙江杭州310006 [2]浙江大学医学院附属儿童医院实验检验中心,浙江杭州310003 [3]浙江大学医学院免疫学研究所,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2013年第4期431-436,共6页Journal of Zhejiang University(Medical Sciences)

摘　　要：目的:考察三尖杉酯碱(HT)对人急性早幼粒白血病(APL)细胞株NB4细胞的生长抑制作用和凋亡诱导作用,并探讨其杀伤白血病细胞的作用机制。方法:采用台盼兰染色法,细胞计数,考察HT对NB4细胞的生长抑制作用和细胞毒性作用;采用AO-EB荧光染色法和细胞形态学观察,考察HT对NB4细胞的凋亡诱导作用;采用PI染色的流式细胞术,考察HT对NB4细胞的凋亡诱导作用;采用Western Blotting技术,考察HT对NB4细胞凋亡相关蛋白的影响;采用siRNA干扰技术,沉默目的蛋白证明推论;提取APL患者血液样本,验证HT的凋亡诱导作用及作用机制。结果:HT能够呈剂量依赖和时间依赖地抑制NB4细胞生长并杀伤NB4细胞,HT处理72h后GI50为(32.0±2.5)nmol/L;HT能够呈现剂量依赖和时间依赖的诱导NB4细胞凋亡,HT处理6h后其诱导凋亡百分率为66%;HT诱导NB4细胞凋亡,引起PARP蛋白裂解,并不下调Bcl-2,不影响Bax和Bak的表达,但显著下调Mcl-1蛋白水平;siRNA沉默Mcl-1的蛋白表达可以显著提高HT对NB4细胞的凋亡诱导作用;HT诱导APL患者白血病样本细胞凋亡,并下调Mcl-1蛋白。结论:HT通过抑制细胞生长和诱导细胞凋亡杀伤NB4细胞,HT可能通过下调Mcl-1蛋白发挥诱导NB4细胞凋亡的抗白血病作用。Objective： To investigate the growth inhibition effect, cytotoxicity and apoptotic induction of harringtonine （HT） in human acute promyelocytic leukemia （APL） NB4 cells, and the related mechanism. Methods： NB4 cells were treated with HT. Total cell numbers were counted by hemocytometer, and cell viabilities were determined by trypan blue exclusion. Apoptotic cells were determined by fluorescence microscopy and FACS after staining with AO and EB or PI, respectively. The cleavage of PARP and the activation of Bax and the expression of anti-apoptotic proteins were determined by Western Blot. siRNA was used to silence the expression of target genes. Primary cells were isolated following Ficoll-Hypaque density gradient centrifugation method. Results： HT inhibited cell growth and induced apoptosis of NB4 cells in a dose- and time-dependent manner. Apoptosis induced by HT was correlated with the down-regulation of Mcl-1 and the cleavage of PARP, while HT did not affect the protein level of Bax and Bak or change the protein level of Bcl-2. The silence of Bcl-XL sensitized HT-induced apoptosis in NB4 ceils. Apoptosis induced by HT in primarily cultured APL cells was also correlated with the down-regulation of Mcl-1. Conclusion. HT inhibits cell growth and induces apoptosis in NB4 cells and primarily cultured APL cells,which may be associated with down-regulation of Mcl-1.

关 键 词：三尖杉酯碱/药理学 白血病 早幼粒细胞 急性/药物疗法 细胞凋亡/药物作用 NB4细胞 MCL-1 

分 类 号：R733.71[医药卫生—肿瘤]