|Budget Amount *help
¥13,600,000 (Direct Cost : ¥13,600,000)
Fiscal Year 1998 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1997 : ¥12,400,000 (Direct Cost : ¥12,400,000)
[Purpose] The aim of this study was to study apoptosis during human thymocyte differentiation and selection.
[Method] Expression of Bcl-2 family (Bcl-2, Bax , Bcl-x) was analyzed by flowcytometry. We studied dexamethasone- or etoposide-induced apoptosis using FACS (exposure of phosphatidylserine on cell surface), electrophoresis of DNA and caspase activity assay, with or without caspase inhibitors.
Then, to investigate positive selection signals, thymocytes were cultured with calcium ionophore (ionomycin) and a protein kinase C (PKC) activator (phorbor 12-myristate l3-acetate PMA), the expression of CD4, CD8 and CD69 was serially examined.
[Results] Expression of Bcl-2 was downregulated at CD4+8+(DP) stage, but CD4+8+69+ thymocytes were Bcl-2 high. Bcl-x was expressed highly on CD4+8+ thymocytes. On the other hand expression of Bax was not different in each stage of thymocyte differentiation. CPP32 activity was not detected upon dexamethasone- or etoposide-induced human thymocyte apoptosis. Caspase inhibitors (DEVD・CHO,YVAD・CHO,Z-VAD・fimk) did not prevent cell surface exposure of phosphatidylserine, a hallmark of apoptosis. Only Z-VAD・fimk prevented DNA fragmentation of dexamethasone- or etoposide-induced apoptosis.
DP thymocytes cultured with a combination of ionomycin and PMA for 20h became CD8SP cells. Expression of CD69, an early activation marker, was high at the end of the culture with ionomycin and PMA.
[Conclusion] l)Bcl-x and Bcl-2 may regulate apoptosis during human thymocyte differentiation coorperatively. 2) Caspase3 may have little role in dexamethasone- and etoposide-induced apoptosis, and another caspase may regulate the apopfosis in the human thymus. 3) Positive selection may require an increase in intracellular Ca^<2+> concentration and PKC activation.