|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1994 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1993 : ¥2,000,000 (Direct Cost : ¥2,000,000)
First, we have purified CD34^+ Cells from bone marrow (BM), peripheral blood (PB) and cord blood (CB) using immunological staining and cell sorting. Based on the expression levels of CD33, CD38, HLA-DR,and c-kit, CD34^+ cells could be subdivided into several populations which represent different stage of differentiation. Then, the interactions between ligands for receptor type tyrosine kinases such as c-kit or flt3 and various cytokines including IL-3, GM-CSF,G-CSF,Epo, IL-9, IL-6, and IL-4 have been extensively studied.
The interactions between these cytokines found in this study are as follows : (1) Until now, IL-3, GM-CSF,SCF,and IL-9 are known as burst-promoting activity (BPA). SCF showed a distinct BPA on CD34^+C-kit^<high> cells, whereas its BPA effect became much weaker when CD34^+C-kit^<low> cells were used as a target. In contrast, IL-9 alone failed to show BPA on CD34^+C-kit^<low> cells. SCF showed distinct synergistic actions with GM-CSF and IL-9 in support of erythroid burst
and/or mixed colony formation. (2) SCF can synergize with G-CSF in support of neutrophil colony formation. SCF showed a distinct synergistic action with IL-4 in neutrophil colony formation when CD34^+c-kit^<low or -> cells were used as targets. (3) IL-4 showed a distinct BPA on CD34^+c-kit^<low> cells. In addition, IL-4 synergized with SCF in support of mixed colony formation. (4) FL did synergize with G-CSF in support of neutrophil colony formation. FL also synergized with IL-3 or GM-CSF in support of mixed colony formation in the presence of Epo. (5) Based on these data, we propose that IL-4 may affect the signal transduction pathways for c-kit/SCF and flt3/FL systems. (6) We established the method which enables large scale production and purification of phosphorothioate type anti-sense oligonucleotides. Antisense TGFbeta induced a increased colony forming ability on CD34^+c-kit^<low> cells, suggesting the release of non-cycling immature o Stem cells into active cell cycle.
We plan to investigate the molecular mechanisms which may explain the cytokine networks we found. Application of antisense DNA technology and differential display PCR method will provide an insight into the cellular basis of hematopoiesis. Less