[Changes in cyclin expression during proliferation and differentiation of CD34(+) cells derived from fetal liver induced by thrombopoietin].
In order to elucidate the intrinsic mechanism underlying proliferation and differentiation of megakaryocytes during ontogenesis, CD34(+) cells were isolated from human fetal liver (FL) with a high-gradient magnetic sorting system (MACS) and were incubated in liquid suspension with 50 and 100 ng/ml of thrombopoietin (TPO) and in MegaCult(Tm) -C semi-solid culture system with 0, 12.5, 25, 50, 100, and 200 ng/ml of TPO. The cell number, colony number of CFU-Mk, platelet-associated antigen phenotype, and DNA ploidy of CD41(+) cells were examined from d 0 to d 12 in culture. The expression patterns of cyclins B1, D1, and D3 were also analyzed by using immunoblot and flow cytometry. TPO stimulated proliferation of CD34(+) cells of FL from 1 x 10(5)/ml to 13.12 +/-4.06 10(5)/ml with 95% of CD41a(+) cells and 3% of CD34(+) cells after 12 d of culture. Most of the megakaryocytes (MKs) derived from FL were in 2 N ploidy class, and few in 4 N ploidy class, but no megakaryocytes ploidy class was higher than 4 N. The effect of TPO on the formation of CFU-Mk colonies from FL derived CD34(+) cells is shown in a dose-response curve. The expression of cyclin B1 increased progressively and the high level of cyclin B1 was maintained in FL CD34(+) cells induced by TPO during 12 d of culture. A high level of cyclin B1 appeared on FL derived MKs of G1 phase at d 12. The expression of cy-of cyclins D1 and D3 gradually increased in FL CD34(+) cells, which was induced by TPO during the initial 6-day incubation. Afterwards, the level of cyclins D1 and D3 decreased progressively, particularly in MKs which were in G2+M phases. These data suggest that (1) TPO induced proliferation and differentiation of FL derived CD34(+) cells through upregulation of cyclin B1 in G2+M phases and cyclins D1 and D3 in all phases of cell cycle, and (2) Continuing high level of cyclin B1 and decreases of cyclins D1 and cyclin D3 on MKs in G2+M phases may contribute to a retardation of MK endoreduplication.
Ma
D C
DC
Northern Hospital, Shenyang 110015. mdc580819@sina.com.cn
Jin
B Q
BQ
Sun
Y H
YH
Chang
K Z
KZ
Dai
B
B
Chu
J J
JJ
Liu
Y G
YG
D018952:Antigens, CD34 / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D016213:Cyclins / Q000378:metabolism*; D005333:Fetus; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D008099:Liver / Q000166:cytology*; D008533:Megakaryocytes / Q000166:cytology*; D013926:Thrombopoietin / Q000494:pharmacology*
D018952:Antigens, CD34; D016213:Cyclins; D013926:Thrombopoietin