RESUMEN
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.
RESUMEN
Objective:To investigate the expression profile of Ly49A in a mouse GVHD model and the ex vivo expression alteration of it in T cells exposal to CsA, IL 4, and IL 15, respectively.Methods:Based on mouse aGVHD model, two color fluorescence flow cytometry was used to assay the expression of Ly49A on splenic and bone marrow CD3 +, CD4 + and CD8 + T cell subsets, before and after engraftment in different time interval. Cell culture was used to analyse in vitro Ly49A expression on splenic T cells treated with IL 4 and IL 15.Results:①In normal B6 and F1 strain, the frequencies of Ly49A + cells in BMCs , splenocytes and PMNCs remained at a low level and almost no, if any, such cells were detected in thymus; ②The proportions of CD3 +/Ly49A + andCD8 +/Ly49A + cells rose in early stage(7th,day) , tended to decline until 5 w post engraftment, and thereafter arose and exceed that pretreatment, which was down regulated in the presence of CsA; ③With or without ConA, the CD3 +/Ly49A + proportion were up modulated in purified CD3 +splenic cells, by IL 4 and IL 15 with no different effect demonstrated between those cytokines.Conclusion:KIR +T lymphocytes were most belong to peripheral lymphocytes. There exist a immunological tolerant tendency between grafts and host in semimatched transplant, and some cytokines and immunosuppressants can enhance the tolerance.