Donahue R E, Kirby M R, Metzger M E, Agricola B A, Sellers S E, Cullis H M
Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20850, USA.
Blood. 1996 Feb 15;87(4):1644-53.
Granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) have been shown to stimulate the circulation of hematopoietic progenitor cells in both mice and nonhuman primates. We evaluated the immunophenotype and cell cycle status of CD34+ cells isolated from the bone marrow (BM) and leukapheresis product of cytokine-mobilized nonhuman primates. CD34+ cells were isolated from rhesus macaques that had received no cytokine therapy, 100 micrograms/kg/d G-CSF, 200 micrograms/kg/d SCF, or a combination of both 100 micrograms/kg/d G-CSF and 200 micrograms/kg/d SCF as a subcutaneous injection for 5 days. BM was aspirated before (day 0) and on the last day (day 5) of cytokine administration. On days 4 and 5, peripheral blood (PB) mononuclear cells were collected using a novel method of leukapheresis. Threefold more PB mononuclear cells were collected from animals receiving G-CSF alone or G-CSF and SCF than from animals that had received either SCF alone or no cytokine therapy. CD34+ cells were positively selected using an immunoadsorptive system from the BM, PB, and/or leukapheresis product. Threefold and 10-fold more CD34+ cells were isolated from the leukapheresis product of animals receiving G-CSF or G-CSF and SCF, respectively, than from animals receiving no cytokine therapy or SCF alone. The isolated CD34+ cells were immunophenotyped using CD34-allophycocyanin, CD38-fluorescein isothiocyanate, and Thy-1-phycoerythrin. These cells were later stained with 4', 6-diamidino-2-phenylindole for simultaneous DNA analysis and immunophenotyping. BM-derived CD34+ cells did not differ significantly in cell cycle status and Thy-1 or CD38 phenotype before or after G-CSF and/or SCF administration. Similarly, CD34+ cells isolated from the leukapheresis product did not differ significantly in immunophenotype or cell cycle status before or after G-CSF and/or SCF administration. However, there were consistent differences in both immunophenotype and cell cycle status between BM- and PB-derived CD34+ cells. CD34+ cells isolated from the PB consistently had a smaller percentage of cells in the S+G2/M phase of the cell cycle and had a higher percentage of cells expressing Thy-1 than did CD34+ cells isolated from the BM. A greater proportion of PB-derived CD34+ cells were in the S+G2/M phase of the cell cycle after culture in media supplemented with interleukin-6 and SCF, However, culturing decreased the proportion of CD34+ cells expressing Thy-1.
粒细胞集落刺激因子（G-CSF）和干细胞因子（SCF）已被证明可刺激小鼠和非人类灵长类动物造血祖细胞的循环。我们评估了从细胞因子动员的非人类灵长类动物的骨髓（BM）和白细胞分离产物中分离出的CD34+细胞的免疫表型和细胞周期状态。从接受过无细胞因子治疗、100微克/千克/天G-CSF、200微克/千克/天SCF或100微克/千克/天G-CSF与200微克/千克/天SCF联合皮下注射5天的恒河猴中分离CD34+细胞。在细胞因子给药前（第0天）和最后一天（第5天）采集骨髓。在第4天和第5天，采用一种新型白细胞分离方法收集外周血（PB）单核细胞。与接受单独SCF或无细胞因子治疗的动物相比，接受单独G-CSF或G-CSF与SCF的动物收集到的PB单核细胞多三倍。使用免疫吸附系统从骨髓、PB和/或白细胞分离产物中阳性选择CD34+细胞。与接受无细胞因子治疗或单独SCF的动物相比，从接受G-CSF或G-CSF与SCF的动物的白细胞分离产物中分离出的CD34+细胞分别多三倍和十倍。使用CD34-别藻蓝蛋白、CD38-异硫氰酸荧光素和Thy-1-藻红蛋白对分离出的CD34+细胞进行免疫表型分析。这些细胞随后用4',6-二脒基-2-苯基吲哚染色，用于同时进行DNA分析和免疫表型分析。在给予G-CSF和/或SCF之前或之后，骨髓来源的CD34+细胞在细胞周期状态以及Thy-1或CD38表型方面没有显著差异。同样，从白细胞分离产物中分离出的CD34+细胞在给予G-CSF和/或SCF之前或之后在免疫表型或细胞周期状态方面也没有显著差异。然而，骨髓来源和PB来源的CD34+细胞在免疫表型和细胞周期状态方面都存在一致的差异。从PB中分离出的CD34+细胞在细胞周期的S+G2/M期的细胞百分比始终较小，并且表达Thy-1的细胞百分比高于从骨髓中分离出的CD34+细胞。在补充白细胞介素-6和SCF的培养基中培养后，更大比例的PB来源的CD34+细胞处于细胞周期的S+G2/M期。然而，培养会降低表达Thy-1的CD34+细胞的比例。
