Fruehauf S, Veldwijk M R, Krämer A, Haas R, Zeller W J
Department of Internal Medicine V, University of Heidelberg, Germany.
Stem Cells. 1998;16(4):271-9. doi: 10.1002/stem.160271.
Treatment with a combination of chemotherapy and G-CSF leads to the release of hematopoietic stem cells from the bone marrow (BM) to the peripheral blood (PB), where they can be harvested for transplantation. Premobilization BM CD34+ cells were reported to proliferate actively, while virtually none of the mobilized PB CD34+ cells were in the S/G2M phase. We were interested in elucidating the cell cycle state further and in investigating the role of adhesion molecule expression on marrow-adherent and circulating CD34+ cells during different phases of the cell cycle. Consecutive premobilization BM and leukapheresis product (LP) samples were obtained from 14 patients following G-CSF-supported chemotherapy. Steady-state BM and LP CD34+ selected cells were triple-stained for CD34, for DNA using the intercalating dye 7-aminoactinomycin D, and for Ki-67, cyclins, or adhesion antigens. Ki-67 is expressed in all phases of the cell cycle except G0 and was found in 69.14%+/-3.46% (mean +/- standard error [SE]) of BM CD34+ cells and 62.78%+/-3.37% of LP CD34+ cells, while in BM significantly more CD34+/Ki-67+ cells were in the S/G2M phase of the cell cycle than in LP (8.6%+/-0.9% versus 1.8%+/-0.3%, respectively, p = 0.0001). Therefore, most circulating mobilized CD34+ cells are in the G1 phase, similar to their steady-state BM counterparts. Cyclin A became detectable in the 2n DNA peak. As expected, a higher proportion of CD34+/cyclin A+/S/G2M cells was found in BM than in LP (p < 0.05). Antigen density of the cyclins D3 and D2 tended to be higher on LP than on BM CD34+ cells, while D1 was found at low levels in similar density. The adhesion antigens CD18, CD49b, CD49d, CD49e, CD58, and CD62L were expressed in a significantly higher proportion of S/G2M-phase than in G0/G1-phase CD34+ cells. The strongest association to the proliferative status was observed for CD49d, which was coexpressed by 85.9% +/-2.6% (BM) or 90.8%+/-2.5% (LP) of CD34+/S/G2M cells, whereas a distinct CD34+/CD49d-/S/G2M population could not be detected. The average coexpression of the other antigens was 57% (CD49e, CD18) or lower. Our results demonstrate that the majority of PB CD34+ cells mobilized following G-CSF-supported chemotherapy and steady-state BM CD34+ cells are in the late G1 phase of the cell cycle and show a correlation between the expression of adhesion receptors and cell cycle status of CD34+ cells in both BM and LP.
化疗与粒细胞集落刺激因子（G-CSF）联合治疗可促使造血干细胞从骨髓（BM）释放至外周血（PB），在此处可采集用于移植。据报道，动员前骨髓CD34+细胞可积极增殖，而动员后的外周血CD34+细胞几乎没有处于S/G2M期的。我们感兴趣的是进一步阐明细胞周期状态，并研究细胞周期不同阶段骨髓黏附及循环CD34+细胞上黏附分子表达的作用。从14例接受G-CSF支持化疗的患者中获取连续的动员前骨髓和白细胞分离产物（LP）样本。对稳态骨髓和LP中经CD34分选的细胞进行三重染色，分别标记CD34、使用插入性染料7-氨基放线菌素D标记DNA以及标记Ki-67、细胞周期蛋白或黏附抗原。Ki-67在细胞周期的所有阶段均有表达，但G0期除外，在骨髓CD34+细胞中占69.14%±3.46%（平均值±标准误[SE]），在外周血LP CD34+细胞中占62.78%±3.37%，而骨髓中处于细胞周期S/G2M期的CD34+/Ki-67+细胞显著多于外周血LP中的（分别为8.6%±0.9%和1.8%±0.3%，p = 0.0001）。因此，大多数循环动员的CD34+细胞处于G1期，与其稳态骨髓对应细胞相似。细胞周期蛋白A在2n DNA峰中可检测到。正如预期的那样，骨髓中CD34+/细胞周期蛋白A+/S/G2M细胞的比例高于外周血LP中的（p < 0.05）。细胞周期蛋白D3和D2在外周血LP CD34+细胞上的抗原密度往往高于骨髓CD34+细胞，而细胞周期蛋白D1在两者中的密度较低且水平相近。黏附抗原CD18、CD49b、CD49d、CD49e、CD58和CD62L在处于S/G2M期的CD34+细胞中的表达比例显著高于处于G0/G1期的。与增殖状态关联最强的是CD49d，在CD34+/S/G2M细胞中，骨髓中有85.9%±2.6%、外周血LP中有90.8%±2.5%共表达CD49d，而未检测到明显的CD34+/CD49d-/S/G2M细胞群体。其他抗原的平均共表达率为57%（CD49e、CD18）或更低。我们的结果表明，G-CSF支持化疗后动员的外周血CD34+细胞以及稳态骨髓CD34+细胞大多处于细胞周期的G1晚期，并且显示出骨髓和外周血LP中CD34+细胞的黏附受体表达与细胞周期状态之间存在相关性。
