【摘要】 目的:探讨B淋巴瘤细胞Syk基因启动子区5′CpG甲基化情况及甲基化抑制剂5-氮杂脱氧胞苷(5-aza-2′-deoxycytidine,5-az-a-CDR)对淋巴瘤细胞的作用机制。方法:采用RT-PCR方法检测人B淋巴瘤细胞株Raji和Ramos中Syk mRNA的表达,用甲基化特异性PCR(MSP)方法检测Syk基因启动子区甲基化情况;采用MTT法检测5-aza-CDR对细胞增殖作用的影响,应用DNA凝胶电泳、流式细胞技术和电镜分析5-aza-CDR对B淋巴瘤细胞增殖、细胞周期和超微结构的影响。结果:Raji细胞Syk基因呈阳性表达,Ramos细胞未检测出Syk mRNA表达。Raji和Ramos细胞中均未检测到Syk基因启动子区的甲基化状态。5-aza-CDR可明显抑制Raji和Ramos细胞增殖,P<0.05;并存在明显的量效和时效依赖关系。5-aza-CDR可诱导Raji和Ramos细胞凋亡,P<0.05。DNA凝胶电泳检测结果显示明显的"DNAladder",并且有明显的细胞凋亡超微结构变化。然而,5-aza-CDR对Raji和Ramos细胞周期未见明显影响。结论:在Raji和Ramos细胞中,Syk基因沉默可能与甲基化无关;5-aza-CDR能明显抑制B淋巴瘤细胞增殖,诱导细胞凋亡,但对细胞周期无明显影响。更多还原

【Abstract】 OBJECTIVE:To investigate the methylation status of 5’CpG in the promoter region of Syk gene of B lymphoma cells,and the mechanism of 5-aza-CDR inhabiting human B lymphoma cells to proliferate.METHODS:Human B lymphoma cells were detected for expression of Syk gene by RT-PCR.Methylation specific PCR was used to detect methylation of Syk promoter region.The proliferation activities of cells were measured by a MTT assay.The cell cycle progression and apoptosis induced by 5-aza-CDR on Raji and Ramos cells were determined by flow cytometry,DNA agarose gel electrophoresis and electron microscopy.RESULTS:The expression of Syk mRNA was detected in Raji cells,but abscent in Ramos cells.No methylation in the Syk gene promoter was found in Raji and Ramos cells.The proliferations of Raji and Ramos were markedly inhibited by 5-aza-CDR in both of dose and time-dependant manner,P<0.05.The apoptosis rates of Raji and Ramos cells were significantly high in the group treated with 5-aza-CDR,P<0.05.The agarose gel electrophoresis showed that there was a clear "DNA ladder" and ultramicrostructure changes of apoptosis,but the cell cycles of Raji and Ramos cells were not obviously altered,P>0.05.CONCLUSIONS:There is no significant correlation between the silencing of Syk gene and gene methylation in Raji and Ramos cells.5-aza-CDR could obviously inhibit the proliferation and augment apoptosis,but not alter the cell cycle.更多还原