神经酰胺在诱导HL-60细胞凋亡中的作用研究

目的：观察神经酰胺对ＨＬ６０细胞凋亡产生的影响、凋亡相关基因ｂｃｌ２变化及与蛋白激酶Ｃ（ＣＰＫ）途径的关系，探讨其在细胞凋亡过程中可能的调节作用。方法：采用ＤＮＡ凝胶电泳、细胞荧光染色、流式细胞术、逆转录聚合酶链反应（ＲＴＰＣＲ）等技术对细胞进行凋亡鉴定及ｂｃｌ２ｍＲＮＡ表达分析。结果：外源性及内源性神经酰胺类药物可诱导ＨＬ６０细胞产生典型的凋亡改变，明显下调ｂｃｌ２ｍＲＮＡ表达，且与刺激药物呈时间、剂量上的依赖性；ＣＰＫ途径激活剂佛波酯（ＰＭＡ）、二酰基甘油单独作用无诱凋作用，但加入内源性ＣＰＫ抑制剂神经鞘氨醇，即可产生凋亡特有的ＤＮＡ梯形降解。结论：神经酰胺类药物诱导的ＨＬ６０细胞凋亡信号，可通过调节抑凋基因ｂｃｌ２表达降低而产生效应，且与ＣＰＫ途径的抑制密切相关

Effect of ceramide in mediating apoptosis in HL-60 cells

Objective:To observe the effect of ceramide on apoptosis in HL60 cells and determine the relationship between the ceramidemediated growth suppression,the expression of bcl2 protoncogene and inhibition of protein kinase pathway.Methods:DNA fragmentation analysis,FACS,fluorescence microscope and RTPCR techniques were employed to determine the apoptosis and bcl2 mRNA levels of HL60 cells.Results:Treatment with cellpermeable and endogenous ceramide in HL60 cells resulted in a time and dosedependent apoptosis and a significant downregulation of bcl2 mRNA expression.Pretreatment with proper dose of phorbol ester (PMA) and diacylglycerol could not induce cell apoptosis but the DNA genome ladders of HL60 cells could be obviously induced following simultaneous use of sphingosine.Conclusions:Ceramide appears to be an important mediator of apoptosis in HL60 cells.The transduction of apoptotic signal may be associated with the downregulation of bcl2 mRNA expression and inhibition of protein kinase pathway.