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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 syndrome (MDS) is a kind of clonal proliferative disease of multipotent hematopoietic stem cells. At present, there is no definite and effective treatment for it. The study found that sodium valproate (VPA) can inhibit the proliferation of tumor cells by inducing cell cycle arrest and apoptosis. This study explored the inhibitory effect of VPA on the proliferation of MUTZ-1 cells and its mechanism. Methods: MTT assay was used to detect the inhibitory effect of VPA on cell proliferation. The morphological changes of VPA were observed by light microscope and electron microscope. Flow cytometry (FCM) The ratio of apoptotic cells and the cell cycle distribution were determined by RT-PCR and Western blotting. The expressions of p21WAF1 (cyclin-dependent kinase inhibitor) Changes in the level of protein expression. Results: VPA inhibited the proliferation of MUTZ-1 cells in a time and concentration-dependent manner. After treated with 4mmol / L LVPA for 48h, the cells showed morphological characteristics of typical apoptotic cells: the apoptotic cells were observed by light microscopy, including pyknosis, pyknosis, nuclear fragmentation and apoptotic bodies. Transmission electron microscopy Visible apoptotic nucleus chromatin edge set, cytoplasm concentration, increased density, visible cytoplasmic irregular size chromatin mass. The results of flow cytometry showed that the apoptotic rates of cells treated with 1,2,4mmol / LVPA for 72h increased from (0.99 ± 0.35)% to (3.14 ± 0.87)%, (14.90 ± 1.04)%, , (22.46 ± 1.74)%, respectively. Compared with the control group, the difference was statistically significant (P <0.05). The proportion of cells in G0 / G1 phase increased gradually and the proportion of S phase cells decreased gradually. P <0.05). The expression of p21WAF1mRNA and p21WAF1 protein in MUTZ-1 cells was significantly increased after VPA treatment for 72h (both P <0.05) by RT-PCR and Western blotting. Conclusion: VPA can inhibit the proliferation and induce the apoptosis of MUTZ-1 cells in the G0 / G1 phase by up-regulating the expression of p21WAF1.