The mechanisms of proliferation and differentiation of hematopoietic multipotent stem cells.

为阐明多能造血干细胞增殖分化的机制，我们在短期悬浮培养中，观察了IL-3、IL-6、干细胞因子、激光诱导因子和促红细胞生成素对早出现集落形成单位S(CFU-S_8)和迟出现集落形成单位S(CFU-S)的影响。采用~3H-TdR掺入法检测IL-3和EPO对巨噬细胞集落形成单位-S的调控是否参与细胞增殖过程。单独加入IL-3或SCF可剂量依赖性地增加S CFU和EPO对CFU-S_8的作用。此外，CFU-S_8和CFU-S_12对IL-3和SCF的量效曲线不同，证实了CFU-S_8和CFU-S_12是相对不同的亚群。包括~3H-TdR自杀在内的结果表明，EPO作用于CFU-S_8和CFU-S_8和CFU-S_12细胞，刺激其增殖并分化为更成熟的群体。IL-3不仅作用于这两个亚群，而且还作用于更原始的对5FU耐药的干细胞群体。对超临界流体作用的详细调查正在进行中。另外两种细胞因子IL-6和LIF与IL-3具有协同刺激活性。这些因素在干细胞增殖调控机制中的生理作用尚不清楚。然而，这些细胞因子对造血干细胞作用的研究是临床应用的重要研究领域，如用于移植的体外扩增。人BFU-E、CFU-GM和CFIJ-Mix通过一步免疫磁珠分离获得了20倍的浓缩。基础研究表明，为了进一步提高临床使用效率，需要进行多步骤分离程序。

To clarify tlie mechanisms of proliferation and differentiation of multipotent hemopoietic stem cells, we investigated the effects of IL-3, IL-6, SCF, LIF and EPO on early appearing CFU-S(CFU-S_8)and late appearing CFU-S(CFU-S_<12>)in short-term suspension culture. We also employed the ^3H-TdR suicide method to test whether the modulation of CFU-S by IL-3 and EPO involved the proliferative process. A single addition of IL-3 or SCF increased the numbers of both subpopulation of CFU-S recovered in culture with dose-dependent fashion, while EPO, at least up to 10u/ml, increased solely CFU-S_8. Further, dose-response curves of CFU-S_8 and CFU-S_<12> to IL-3 and SCF were different, which confirmed the speculation that CFU-S_8 and CFU-S_<12> are relatively different subpopulation. Our results including ^3H-TdR suicide suggested that EPO acted on both of CFU-S_8 and CFU-S_<12> to stimulate proliferation with differentiation into more mature population in erythroi. d lineage, and that IL-3 acted not only on these two subpopulation but also on more primitive stem cell population which was resistant to 5FU. The investigation on ihe action of SCF in detail is on going. Another two cytokines, IL-6 and LIF, showed synergistic stimulating activity with IL--3. The physiological roles of these factors in the regulatory mechanisms of stem cell proliferation are still unknown. However, studies of tlie action of these cytokines on hemopoietic stem cells are important research fields in regarding to clinical uses such as in votro expansion for transplantation.Human BFU-E, CFU-GM, and CFIJ-Mix were 20x enriched by one-step immunomagnetic beads separation using anti-CD_<34> antibody. Basical studies suggested multi-step separation procedures should be required for further improvement of the efficiency for clinical use.