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最近 ,克隆了一种新的选择性定位于线粒体的神经酰胺酶 ,提示线粒体存在着神经酰胺代谢途径 ,可能对线粒体的功能 ,特别对凋亡产生影响。本研究将线粒体神经酰胺酶基因转染到K5 6 2细胞 ,以此了解线粒体神经酰胺酶的细胞生物学效应。以脂质体介导将含有线粒体神经酰胺酶cDNA的pcDNA3.1 His CDase质粒转染到K5 6 2细胞 ,G4 18筛选阳性克隆 ,建立稳定表达线粒体神经酰胺酶的K5 6 2TC细胞株。用MTT法、annexinⅤ PI法、FCM及Westernblot分别检测K5 6 2与K5 6 2TC细胞在细胞毒药物抗性、血清剥夺耐受性及Bcl 2蛋白表达水平方面的差异。结果表明 :虽然K5 6 2TC与K5 6 2细胞对阿霉素、足叶乙甙及亚砷酸的敏感性没有差异 ,但是K5 6 2TC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K5 6 2TC细胞线粒体神经酰胺酶 ,下调Bcl 2蛋白表达水平 ;二甲基鞘氨醇 (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K5 6 2TC细胞Bcl 2蛋白表达水平 ,而 1 磷酸鞘氨醇明显上调K5 6 2细胞Bcl 2表达水平。结论 :线粒体神经酰胺酶将线粒体神经酰胺代谢为鞘氨醇 ,进而在鞘氨醇激酶作用下生成 1 磷酸鞘氨醇 ,此代谢途径上调K5 6 2细胞Bcl 2蛋白表达水平。
Recently, a new, selective mitochondrial-targeting neuraminidase was cloned, suggesting that mitochondria present a ceramide metabolic pathway that may have an impact on mitochondrial function, particularly apoptosis. In this study, mitochondrial neuramidase gene was transfected into K562 cells in order to understand the cellular biological effects of mitochondrial neuramidase. The pcDNA3.1 His CDase plasmid containing mitochondrial ceramidase cDNA was transfected into K562 cells by lipofectamine and the positive clones were screened by G418 to establish a K5 6 2TC cell line stably expressing mitochondrial ceramidase. The differences of cytotoxicity, serum deprivation tolerance and Bcl 2 protein expression between K562 and K5 6 2TC cells were detected by MTT, annexin Ⅴ PI, FCM and Western blot respectively. The results showed that although the sensitivity of K5 6 2TC and K5 6 2 cells to doxorubicin, etoposide and arsenious acid was not different, the level of Bcl 2 protein in K5 6 2TC cells was significantly increased and the antiserum deprivation ability was significantly enhanced . The antisense oligonucleotide of thio was used to specifically block the mitochondrial ceramidase of K5 6 2TC cells and down-regulate the expression of Bcl 2 protein. Dimethyl sphingosine (a sphingosine kinase inhibitor, reduced intracellular sphingosine-1-phosphate Level) also down-regulated Bcl 2 protein expression in K5 6 2TC cells, whereas sphingosine-1 phosphate significantly up-regulated Bcl 2 expression in K5 6 2 cells. CONCLUSION: Mitochondrial ceramide metabolizes mitochondrial ceramide into sphingosine, which in turn generates sphingosine-1-phosphate by sphingosine kinase. This metabolic pathway upregulates Bcl-2 protein expression in K562 cells.