丙戊酸钠对骨髓增生异常综合征细胞株MUTZ-1增殖和凋亡的影响

Effects of Sodium Valproate on Proliferation and Apoptosis of Human Myelodysplastic Syndromes Cell Line MUTZ-1

背景与目的:骨髓增生异常综合征(myelodysplastic syndrome,MDS)是一类多能造血干细胞的克隆增殖性疾病,目前临床尚无明确有效的治疗方法。研究发现丙戊酸钠(sodium valproate,VPA)可通过诱导细胞周期阻滞和凋亡来抑制肿瘤细胞的增殖,本实验探讨VPA对MDS细胞MUTZ-1增殖的抑制作用及其作用机理。方法:采用四甲基偶氮唑蓝(MTT)比色法检测VPA对细胞增殖的抑制作用;采用光学显微镜和电子显微镜观察VPA作用后细胞形态的变化;应用流式细胞术(FCM)检测不同浓度药物作用后细胞凋亡的比例及细胞周期分布的变化;通过逆转录聚合酶链反应(RT-PCR)技术和Westernblot方法分别检测药物作用后p21WAF1(细胞周期依赖性激酶抑制因子)在mRNA和蛋白质水平表达量的改变。结果:VPA对MUTZ-1细胞的增殖抑制作用呈时间和浓度依赖性。经4mmol/LVPA处理MUTZ-1细胞72h后,细胞呈现典型的凋亡细胞的形态特征:光镜下可见凋亡细胞胞体固缩、核固缩、核碎裂及凋亡小体;透射电镜下可见凋亡细胞核染色质边集、胞浆浓缩、密度增加,胞浆内可见大小不规则的染色质团块。流式细胞术检测结果表明经1、2、4mmol/LVPA作用72h后,细胞的凋亡率由处理前的(0.99±0.35)%分别上升为(3.14±0.87)%、(14.90±1.04)%、(22.46±1.74)%,与对照组相比差异有统计学意义(P<0.05);G0/G1期细胞比例逐渐增多,S期细胞比例逐渐减低,细胞被阻滞在G0/G1期(P<0.05)。RT-PCR和Westernblot技术均发现VPA作用MUTZ-1细胞72h后,明显促进p21WAF1mRNA和p21WAF1蛋白的表达(P<0.05)。结论:VPA能够通过上调p21WAF1的表达,阻滞MUTZ-1细胞于G0/G1期,最终抑制肿瘤细胞的增殖并诱导其凋亡。

BACKGROUND ＆ OBJECTIVE： Myelodysplastic syndromes （MDS） are heterogeneous disorders with the expansion of malignant clones. No satisfactory treatment for MDS is available. Sodium vaiproate （VPA） can inhibit the proliferation of tumor cells by inducing Go/G1 phase arrest and cell apoptosis. This study was to investigate the effects of VPA on the proliferation of MDS cell line MUTZ-1, and explore possible mechanisms. METHODS. MUTZ-1 cells were treated with VPA. Cell proliferation was determined by MTT assay. Cell morphology was observed under microscope and transmission electron microscope, Cell apoptosis and cell cycle were analyzed by flow cytometry （FCM）. The expression of p21^WAF1 （cyclin-dependent kinase inhibitor） was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. RESULTS. VPA inhibited the proliferation of MUTZ-1 cells in concentration- and time-dependent manners, When treated with 4 mmol/L VPA for 72 h, typical apoptotic morphologic features appeared in MUTZ-1 cells, condensation of cells and nuclear chromatin, disintegration of nuclear chromatin, and apoptotic bodies were observed under microscope; aggregation and margination of apoptotic nuclear chromatin, cytoplasm condensation, and irregular chromatin masses were observed under transmission electron microscope. The apoptosis rate was significantly higher in the cells treated with 1, 2, and 4 mmol/L VPA for 72 h than in untreated cells [（3.14±0.87）%, （14.90±1.04）% and （22.46±1.74）% vs, （0.99±0.35）%, P〈0.05]. After treatment of VPA, cell cycle was arrested obviously at G0/G1 phase （P〈0.05）; the mRNA and protein levels of p21^WAF1 were up-regulated in MUTZ-1 cells （P〈0.05）. CONCLUSIONS. VPA could induce G0/G1 phase arrest, inhibit the proliferation and induce the apoptosis of MUTZ-1 cells in vitro. The mechanism may be associated with the upregulation of p21^WAF1.