摘要
目的探讨甲基化抑制剂5-氮杂-2'-脱氧胞苷(5-aza-2'-deoxycytidine,5-aza-CdR)对人急性淋巴细胞白血病细胞株CEM中抑癌基因EphB4的转录调控作用及对CEM细胞增殖凋亡的生物学影响,为寻找肿瘤治疗新靶点提供实验依据。方法采用亚硫酸氢盐修饰后测序法检测EphB4基因甲基化水平,荧光定量PCR法(Q-PCR)和Western blot方法检测5-aza-CdR处理前后EphB4基因mRNA及蛋白表达水平;MTS法检测不同剂量(1.0、2.5、5.0μmol/L)的5-aza-CdR作用于CEM细胞0 h、24 h、48 h、72 h、96 h对CEM细胞存活率的影响,流式细胞仪分析5-aza-CdR作用96 h对CEM细胞周期及凋亡的影响。结果 CEM细胞中存在EphB4基因的甲基化,甲基化水平为31.4%;不同浓度5-aza-CdR作用后CEM细胞甲基化水平均下降。5-aza-CdR作用使CEM细胞中EphB4基因的mRNA和蛋白表达上升;5-aza-CdR明显抑制CEM细胞增殖,并与药物浓度及作用时间呈正相关。5-aza-CdR处理96 h后,早期凋亡由4.1%上升为24.8%。用药96 h后,CEM细胞G1期由62.4%降至46.8%,G2期由2.1%升至16.2%,细胞阻滞于G2期。结论特异性甲基化转移酶抑制剂5-aza-CdR能使CEM细胞中沉默的EphB4基因重新表达,并可抑制白血病细胞增殖和诱导其凋亡。
Objective To study the regulation of methylation inhibitor 5-aza-2′-deoxycytidine on transcription of EphB4 gene and effects on the proliferation and apoptosis of human acute lymphocyte leukemia cell line CEM. Methods Bisulfite sequencing PCR was used to detect CpG island methylation density in EphB4 promoter.The expression of EphB4 mRNA and protein was determined by Q-PCR and Western blot.MTS assay and flow cytometry were used to detect the apoptosis of CEM cells after treatment with different concentrations of 5-aza-2′-deoxycytidine（1.0,2.5 and 5 μmol/L）. Results Methylation of EphB4 gene promoter was detected in CEM cells（31.4%）.The methylation level of EphB4 gene was down-regulated after treatment with various concentrations of 5-aza-2′-deoxycytidine.The EphB4 mRNA and protein expression in CEM cells increased after 5-aza-2′-deoxycytidine treatment.5-Aza-2′-deoxycytidine significantly inhibited the cell growth in dose and time dependent manners.Early apoptosis rates of CEM cells increased from 4.1% to 24.8% 96 hrs after 5-aza-2′-deoxycytidine treatment.CEM cells in G1 phase decreased from 62.4% to 46.8%,cells in G2 phase increased from 2.1% to 16.2%,and CEM cells were arrested in G2 phase after treatment with 5 μmol/L 5-aza-2′-deoxycytidine for 96 hrs. Conclusions 5-Aza-2′-deoxycytidine,an inhibitor of specific methylation transferase,can induce expression of the silent EphB4 gene in CEM cells,inhibit the proliferation of leukemia cells and induce cell apoptosis.
出处
《中国当代儿科杂志》
CAS
CSCD
北大核心
2012年第3期205-209,共5页
Chinese Journal of Contemporary Pediatrics
基金
广东省自然科学基金(No.S2011010004349)
深圳市科技计划项目(No.201102156)
