【摘要】 目的:将线粒体神经酰胺酶(mtCDase)转染到K562细胞,观察线粒体神经酰胺酶的细胞生物学效应。方法:以脂质体介导将含有mtCDase cDNA的pCDNA3·1/His-CDase质粒转染到K562细胞,G418筛选阳性克隆,建立稳定表达mtCDase的K562TC细胞株。AnnexinⅤ/PI法、FCM及Western印迹法分别检测K562与K562TC细胞在无血清培养耐受性及Bcl-2蛋白表达水平方面的差异。结果:稳定表达mtCDase的K562TC细胞Bcl-2蛋白水平明显升高,抗血清剥夺能力明显增强。以硫代反义寡核苷酸特异性封闭K562TC细胞mtCDase,Bcl-2蛋白表达水平下调;二甲基鞘氨醇(DMS,鞘氨醇激酶抑制剂,降低细胞内1-磷酸鞘氨醇水平)同样下调K562TC细胞Bcl-2蛋白表达水平,而外源性1-磷酸鞘氨醇(SPP)显著上调K562细胞Bcl-2表达水平。结论:mtCDase转染K562细胞导致Bcl-2蛋白表达水平升高及无血清培养耐受能力增强。这种效应是通过mtCDase的下游代谢产物-SPP产生的。本研究证实mtCDase通过其下游代谢产物代谢SPP,上调K562细胞Bcl-2蛋白表达水平。更多还原

【Abstract】 AIM: To investigate the role of mitochondrial ceramidase in mitochondrial functions, especially in the regulation of apoptosis. METHODS: pCDNA3.1/His-MtCDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transfected into K562 cells by liposome, and G418 was used to screen the positive clones. A stable transfected K562 cell line was established and defined as ‘K562TC’. The differences between K562 and K562TC cells in serum withdrawal resistance and Bcl-2 protein expression were evaluated by annexin V/PI test, flow cytometry and Western blotting, respectively. RESULTS: K562TC cells with elevated Bcl-2 protein expression level identified by FCM or Western blotting showed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N’-dimethylsphingosine (DMS), a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate (SPP) production, also abrogated Bcl-2 protein expression in K562TC cells, while exogenous sphingosine-1-phosphate up-regulated Bcl-2 protein level in K562 cells. CONCLUSION: Mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form, indicating that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.更多还原