【摘要】 目的探讨脱乙酰化酶抑制剂丁酸钠(SB)与去甲基化制剂5-氮杂-2’-脱氧胞苷(5-Aza-CdR)联合处理经骨髓瘤细胞系U266诱导高甲基化失活的p16基因重新表达的可能性及其对细胞生长的影响。方法用不同浓度药物处理U266细胞后测定细胞生长曲线;流式细胞术分析细胞周期;RT-PCR和Westernblotting检测p16基因mRNA及其蛋白的表达水平。结果单用5-Aza-CdR或SB均抑制细胞生长,联合用药对细胞的抑制作用明显增强;单用5-Aza-CdR或SB对细胞G1期无影响,SB与5-Aza-CdR联合作用发生显著的G1期阻滞;单用0.1μmol/L5-Aza-CdR即可诱导U266细胞p16基因重新表达,随着5-Aza-CdR浓度的增高,p16表达增加,单用SB只能诱导p16基因的弱表达,联合用药明显增强p16基因表达。结论脱乙酰化酶抑制剂SB与去甲基化制剂5-Aza-CdR协同可显著诱导人骨髓瘤细胞U266因高甲基化失活的p16基因重新表达,细胞生长受抑,并使细胞阻滞在G期。更多还原

【Abstract】 Objective To understand the synergic effect of histone deacetylase i nhibitor sodium butyrate (SB) and demethylat-ing agent 5-Aza-2’-deoxycytidine ( 5-Aza-CdR) on cell growth and to explore the possibility of re-expression of the hyperme-thylated and silenced p16 gene in the myeloma cell line U266. Methods U 266 cells were cultured in RPMI 1640 in the pres-ence of varied doses of SB and 5-Aza-CdR, and the growth curve was obtained by trypan-blue exclusion assay and cell count. The cell cycle was analyzed by flow cytometry and the expression le vel of mRNA and protein of p16 gene were detected by reverse transcriptase-PCR and Western blotting, respectively. Results The cell growth was arrested by trea tment with 5-Aza-CdR alone or SB alone. Increased inhibition effect was shown i n synergic treratment of SB and 5-Aza-CdR. The G 1 phase of cell cycle was arrested by 5-Aza-CdR combined with SB, which, how ever, did not occur when SB or 5-Aza-CdR was used alone. 5-Aza-CdR alone induced the expression of p16 gene in a concentration-dependent manner, whereas SB alon e only induced its low-level expression. The expression level of both mRNA and protein of p16 gene was increased significantly by synergic ap-plication of SB a nd 5-Aza-CdR. Conclusions Hypermethylated and silenced p16 gene in U266 cell lin e can be markedly reac-tivated by synergic treatment with demethylating agent 5-Aza-CdR and histone deacetylase inhibitor SB, and the cell growth can be inhibi ted and cell cycle arrested at G 1 phase.更多还原