去甲基药5-氮杂胞苷对Caspase-8缺乏的神经母细胞瘤细胞化疗敏感性的影响  

Influence of 5-Azacytidine on Drug Resistance of Caspase-8 Silence Neuroblastoma

作　　者：周广玉[1] 李爱敏[1] 初清[1] 王晓莉[1] 

机构地区：[1]青岛大学医学院附属烟台毓璜顶医院儿科,山东烟台264000

出　　处：《实用儿科临床杂志》2010年第15期1158-1160,共3页Journal of Applied Clinical Pediatrics

基　　金：烟台市科学技术计划项目(2004221)

摘　　要：目的探讨去甲基药5-氮杂胞苷能否恢复神经母细胞瘤(NB)细胞中Caspase-8的表达水平,从而提高NB细胞SY5Y对常用化疗药物依托泊苷的敏感性。方法采用反转录-PCR(RT-PCR)技术检测5-氮杂胞苷作用前后Caspase-8mRNA的表达。Elivi-sion二步法检测5-氮杂胞苷作用前后SY5Y细胞中Caspase-8蛋白的表达。应用四甲基偶氮唑蓝(MTT)法检测Caspase-8缺乏的NB细胞中加入化疗药依托泊苷后细胞存活率,比较加入5-氮杂胞苷后再加入依托泊苷后细胞存活率的变化。结果 RT-PCR方法未检测到SY5Y细胞中Caspase-8mRNA表达,5-氮杂胞苷单独作用3d可以检测到Caspase-8mRNA表达,5d组Caspase-8mRNA表达较3d组明显增强,且Caspase-8mRNA表达水平随5-氮杂胞苷水平增高而增高。Elivision二步法检测SY5Y细胞中Caspase-8蛋白表达阴性,5-氮杂胞苷作用3d后Caspase-8蛋白表达呈阳性。MTT法检测发现不同质量浓度的依托泊苷(0.1、0.3、1.0、2.5)mg.L-1单独作用24h组细胞存活率分别为94.16%±5.89%、76.46%±5.83%、44.31%±5.05%、36.15%±5.71%;5-氮杂胞苷+同质量浓度依托泊苷组细胞存活率分别为84.23%±6.95%,61.00%±4.98%,31.64%±3.84%,23.75%±1.89%,联合作用组细胞存活率较单独作用组细胞存活率明显下降,其差异均有统计学意义(Pa<0.01)。结论 SY5Y细胞不表达Caspase-8mRNA及蛋白,5-氮杂胞苷可诱导SY5Y细胞Caspase-8mRNA及蛋白表达,且随时间延长,Caspase-8mRNA表达随之增加。依托泊苷对NB细胞SY5Y具有增殖抑制作用,5-氮杂胞苷可增强其对SY5Y的增殖抑制作用,并提高化疗的敏感性。Objective To approach the effect of 5-azacytidine,a DNA methylation inhibitor on the Caspase-8 expression and to explore whether restoration of Caspase-8 would improve chemotherapeutic agent etoposide sensitivity to neuroblastoma（NB） cell line SY5Y.Methods The level of Caspase-8 mRNA before and after using 5-azacytidine was detected by RT-PCR.The expression of Caspase-8 protein in NB cell line SY5Y before and after using 5-azacytidine was detected by immunochemistry.The changes of cell survival rate of different concentration of etoposide alone,etoposide＋5-azacytidine was detected by methy thiazolyl tetrazolium（MTT） method.Results Expression of Caspase-8 mRNA was undetectable in SY5Y cell line by the methods of RT-PCR,but the expression of Caspase-8 mRNA was detectable after treatment with 5-azacy-tidine for 3 days,and an increased expression of Caspase-8 was found after treatment with 5-azacytidine for 5 days.The expression of Caspase-8 protein was undetectable in SY5Y,it was positive after treatment of 5-azacytidine for 3 days by cell immunochemistry.The survival rates of different concentration of etoposide alone to NB cell for 24 h （0.1,0.3,1.0,2.5 mg·L^-1） were 94.16%±5.89%,76.46%±5.83%,44.31%±5.05%,36.15%±5.71%;the survival rates of different concentration of etoposide＋5-azacytidine were 84.23%±6.95%,61.00%±4.98%,31.64%±3.84%,23.75%±1.89%.The survival rates of 5-azacytidine combined with different concentration of etoposide were significantly lower than that of etoposide alone （Pa0.01）.Conclusions Chemotherapeutic agent etoposide has anti-proliferation effect on the SY5Y cells,5-azacytidine can increase this effect,the combination treatment of 5-azacytidine and etoposide may greatly enhance the killing rates of etoposide to the SY5Y cells.

关 键 词：神经母细胞瘤 半胱-天冬氨酸蛋白酶-8 5-氮杂胞苷 肿瘤 

分 类 号：R739.41[医药卫生—肿瘤]