血小板生成素诱导胎肝CD34^+造血干/祖细胞增殖分化与周期蛋白表达的关系  

Changes in cyclin expression during proliferation and differentiation of CD34^+ cells derived from fetal liver induced by thrombopoietin

作　　者：马东初[1] 金伯泉[2] 孙英慧[1] 常奎忠[1] 戴兵[1] 初俊杰[1] 刘亚革[1] 

机构地区：[1]沈阳军区总医院医学实验科,沈阳110015 [2]第四军医大学免疫教研室,西安710032

出　　处：《生理学报》2001年第4期296-302,共7页Acta Physiologica Sinica

基　　金：theMedicalandPharmaceuticalFoundationofPLA (No b96D0 0 3)

摘　　要：为了解胚胎时期巨核细胞增殖分化特有的内在机制 ,本研究观察了在体外培养体系中 ,胎肝源CD3 4 +造血干 /祖细胞在血小板生成素 (thrombopoietin ,TPO)作用下增殖分化特征与相关周期蛋白B1、D1和D3表达及细胞内水平变化的关系。结果发现 :( 1)经 12d培养后 ,TPO使胎肝源CD3 4 +细胞数从 1× 0 5个细胞 /ml增加到 13 12± 4 0 6× 10 5个细胞 /ml,CD4 1+细胞增加到了 95 % ,CD3 4 +细胞下降到了 3 % ,大部分细胞的DNA倍性为 2N ,少数为 4N ,无大于 4N巨核细胞 ,TPO对MegaCultTm C胶原半固体培养体系中胎肝源CD3 4 +细胞形成CFU Mk集落产率的影响呈明显的剂量效应关系 ;( 2 )在整个培养期间 ,周期蛋白B1表达逐渐增加 ,并保持在一个高水平上 ,培养后期 ,高水平的周期蛋白B1出现在G1期细胞上 ;( 3 )周期蛋白D1和D3表达先增加 ,培养后期细胞内水平下降 ,且以G2期细胞为主。该结果提示 :( 1)TPO通过上调周期蛋白B1和在所有细胞周期时限上调周期蛋白D1和D3表达 ,促进巨核细胞祖细胞的增殖分化 ;( 2 )周期蛋白B1在G2 +M期的持续高水平和周期蛋白D1和D3在G2 +M期的水平下降 ,可能导致胎肝源巨核细胞核内有丝分裂延迟或阻滞。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 1×10 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 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.

关 键 词：周期蛋白 血小板生成素 CD34^+细胞 巨核细胞 造血干细胞 祖细胞 胎肝 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学] Q461[医药卫生—基础医学]