地西他滨联合丙戊酸钠诱导的骨髓瘤细胞凋亡及其机制研究  

Induction effects of decitabine in combination with valproic acid sodium on apoptosis of myeloma cells and its underlying mechanism

作　　者：马泳泳[1] 周淑娟[1] 陈枫煜[2] 蔡芳芳[1] 俞康[1] 

机构地区：[1]温州医学院附属第一医院血液科,浙江温州325000 [2]温州医学院第一临床医学院,浙江温州325000

出　　处：《解放军医学杂志》2013年第10期837-841,共5页Medical Journal of Chinese People's Liberation Army

基　　金：温州市科技计划(Y20100106)~~

摘　　要：目的探讨地西他滨(DCA)联合丙戊酸钠(VPA)体外诱导骨髓瘤细胞株RPMI 8226凋亡及对死亡相关蛋白激酶(DAPK)基因去甲基化的作用。方法体外培养多发性骨髓瘤RPMI 8226细胞,实验分为5组:DCA组(选取1.5、3μmol/L 2个浓度),VPA组(选取1、2μmol/L 2个浓度),DCA+VPA组(DCA 3μmol/L+VPA 2μmol/L),PBS组,阴性对照组。上述细胞分别培养24、72h后,MTT法检测细胞存活情况。培养72h后,透射电镜观察DCA+VPA组细胞凋亡形态;流式细胞仪Annexin V法检测上述5组细胞凋亡率;MSP法检测上述5组细胞(DCA组取3μmol/L浓度,VPA组取2μmol/L浓度)处理前后DAPK基因甲基化状态,半定量RT-PCR方法检测DAPK基因表达情况。结果处理72h后,DCA+VPA组MTT值下降最明显,细胞存活率降低,与PBS组及阴性对照组比较差异有统计学意义(P<0.05);VPA组及DCA组MTT值均下降,DCA 3μmol/L组与PBS组及阴性对照组比较差异有统计学意义(P<0.05)。处理72h后,DCA+VPA、VPA、DCA组G1/G0期细胞增多,S期细胞减少,细胞凋亡率增加,DCA+VPA组凋亡率高于DCA组及VPA组(P<0.05),与PBS组及阴性对照组比较差异亦有统计学意义(P<0.05)。透射电镜下可观察到DCA+VPA组细胞出现核染色质凝聚、固缩、边集、核碎裂等典型的细胞凋亡形态学改变。处理72h后,DCA+VPA、VPA、DCA组细胞DAPK启动子均有去甲基化表现,以DCA+VPA组去甲基化程度为最大,与PBS组及阴性对照组相比差异均有统计学意义(P<0.05)。DCA+VPA、VPA、DCA组DAPK基因mRNA表达量均增加,且DCA+VPA组的相对mRNA比值高于其余4组(P<0.05)。结论DCA联合VPA可诱导骨髓瘤细胞株RPMI 8226凋亡及DAPK基因启动子去甲基化,使DAPK基因表达恢复。Objective To investigate the effects of decitabine （DCA） combined with valproate sodium （VPA） on the apoptosis of multiple myeloma cell line RPMI 8226 and the demethylation of death-associated protein kinase （DAPK）. Methods The RPMI 8226 cells were cultured and treated with DCA （1.5μmol/L, 3μmol/L）, VPA （1μmol/L, 2μmol/L）, VPA＋DCA （DCA 3μmol/L＋VPA 2μmol/L） and PBS, respectively, and those cells without treatment served as negative control. The survival status of cells was detected by MTT assay after culture for 24 and 72h. The apoptotic rate and cell cycle of RPMI 8226 cells were detected by flow cytometry （FCM）, and the apoptosis of cell was observed under the transmission electron microscope （TEM）. MSP method was used to detect the methylation of DAPK gene in the RPMI 8226 cells. The mRNA expression of DAPK was detected by RT- PCR. Results MTT assay showed that the cell activity decreased remarkably in VPA＋DCA group after 72h compared with other groups, and the MTT value of VPA＋DCA group was significantly different from those of PBS and negative control group （P〈0.05）. The cell activity in VPA and DCA group decreased remarkably, the MTT value of DCA 3μmol/L group was significantly different from those of PBS and negative control group （P〈0.05）. FCM analysis revealed that the apoptosis rate increased, the cells in G0/G~ phase increased and the cells in S phase decreased in VPA, DCA and VPA＋DCA groups after 72h. The apoptosis rate was higher in DCA＋VPA group than in DCA and VPA groups, and significantly higher than in PBS and negative control groups （P〈0.05）. The typical morphological changes of RPMI 8226 cells were observed under TEM in DCA＋VPA group, such as chromatin condensation, pyknosis, margination and nuclear fragmentation. The demethylation of DAPK promoters was observed in all the VPA, DCA and VPA＋DCA groups, with a highest degree in DCA＋VPA group. The degree of DAPK demethylation was significantly different in DCA＋VPA group fr

关 键 词：健康行为 健康因素 糖尿病 C反应蛋白 

分 类 号：R551.31[医药卫生—血液循环系统疾病]