MATSUYAMA Shusuke Yokohama City University, School of Medicine, Professor, 医学部, 教授 (20045983)
FUNABIKI Tetsunori Yokohama City University, School of Medicine, Instructor, 医学部, 助手 (20264616)
IKUTA Koichiro Yokohama City University, School of Medicine, Assistant Professor, 医学部, 講師 (80159590)
甲斐 純夫 横浜市立大学, 医学部, 助手 (10233644)
井上 達 横浜市立大学, 医学部, 助教授 (50100110)
|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1994 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
We examined the effects of cytokines on the growth of murine megakaryocytic cell lines, L8057 and L8057Y5, which had been maintained in protein-free culture. In serum-free methylcellulose culture, the addition of rhEPO,rmIL-3, rhIL-6, rmLIF,or rmGM-CSF enhanced colony growth of L8057Y5 cells mostly in dose-dependent fashion Also, colony growth of L8057 cells was enhanced by these cytokines, but to a lesser extent. The expression of mRNAs for EPO-R,IL-6R,gp130, IL-3R subunits were constitutively expressed in both lines. We also tested the effect of rmTpo, which was newly cloned in 1994, on the megakaryocyte progenitors in marrow. The megakaryocyte colony growth was observed by the stimulation of sole rmTpo. The addition of rmIL-3 with rmTpo showed increase not only in the total number, but in the frequencies of large colonies and of mixed-type colonies. Also, rhEpo and GM-CSF showed synergistic effects with rmTpo on the colony formation. Colony formation by BFU-Es and CFU-GMs was not significantly reduced by the addition of rhTGF-beta_1 ; however, the growth of CFU-Mks, stimulated by rmIL-3, rmTPO,or both cytokines, was markedly inhibited, which suggest that TGF-beta is a potent negative-regulator in megakaryocytepoiesis.
rmTpo also enhanced the growth of L8057 cells mostly in a concentration-dependent fashon as observed in the growth of CFU-Mk. RT-PCR revealed the costitutive expression of Mpl-mRNA in L8057 cells. These results suggest that L8057 and L8057Y5 cell lines have the characteristics of megakaryoblastic cells in their biological responses to cytokines, and that the growth-enhancing effects of these cytokines on the cell lines may be achieved through specific receptors. For totally clarifying the regulation mechanisms of thrombopoiesis, it should be promissing way to investigate signaling cascades for every cytokines including Tpo in megakaryoblastic cells. Again our findings validate the usefulness of L8057 cells for these purposes.