Lewis John L, Marley Stephen B, Ojo Morenike, Gordon Myrtle Y
LRF Centre for Adult Leukaemia, Department of Hematology, Faculty of Medicine, Imperial College, London, England, UK.
Exp Hematol. 2004 Jan;32(1):36-44. doi: 10.1016/j.exphem.2003.09.016.
To investigate 1) the effects of lineage-specific cytokines (G-CSF and EPO) combined with ligands for different classes of cytokine receptors (common beta chain, gp130, and tyrosine kinase) on proliferation by human myeloid and erythroid progenitor cells; and 2) the signal transduction pathways associated with combinatorial cytokine actions.
CFU-GM and BFU-E were cloned in vitro. Secondary colony formation by replated CFU-GM and subcolony formation by BFU-E provided measures of progenitor cell proliferation. Studies were performed in the presence of cytokine combinations with and without signal transduction inhibitors.
Proliferation by CFU-GM and BFU-E was enhanced synergistically when common beta chain receptor cytokines (IL-3 or GM-CSF) were combined with G-CSF or EPO, but not with gp130 receptor cytokines (LIF or IL-6) or tyrosine kinase receptor cytokines (SCF, HGF, Flt-3 ligand, or PDGF). Delayed addition studies with G-CSF+IL-3 and EPO+IL-3 demonstrated that synergy required the presence of both cytokines from the initiation of the culture. The Jak2-specific inhibitor, AG490, abrogated the effect of combining IL-3 with EPO but had no effect on the enhanced CFU-GM proliferation stimulated by IL-3+G-CSF. The PI3 kinase inhibitors LY294002 and wortmannin substituted for G-CSF in combination with IL-3 since proliferation in the presence of LY294002/wortmannin+IL-3 was enhanced to the same extent as in the presence of G-CSF+IL-3. In contrast, LY294002 and wortmannin inhibited proliferation in the presence of EPO and in the presence of EPO+IL-3.
1）研究谱系特异性细胞因子（粒细胞集落刺激因子和促红细胞生成素）与不同类细胞因子受体（共同β链、糖蛋白130和酪氨酸激酶）的配体联合应用对人髓系和红系祖细胞增殖的影响；2）研究与细胞因子联合作用相关的信号转导途径。
体外克隆粒-巨噬细胞集落形成单位（CFU-GM）和爆式红系集落形成单位（BFU-E）。通过重铺CFU-GM形成次级集落以及BFU-E形成亚集落来衡量祖细胞增殖。在有或无信号转导抑制剂的细胞因子组合存在的情况下进行研究。
当共同β链受体细胞因子（白细胞介素-3或粒细胞-巨噬细胞集落刺激因子）与粒细胞集落刺激因子或促红细胞生成素联合时，CFU-GM和BFU-E的增殖协同增强，但与糖蛋白130受体细胞因子（白血病抑制因子或白细胞介素-6）或酪氨酸激酶受体细胞因子（干细胞因子、肝细胞生长因子、Flt-3配体或血小板衍生生长因子）联合时则不然。粒细胞集落刺激因子+白细胞介素-3和促红细胞生成素+白细胞介素-3的延迟添加研究表明，协同作用需要从培养开始就同时存在两种细胞因子。Jak2特异性抑制剂AG490消除了白细胞介素-3与促红细胞生成素联合的作用，但对白细胞介素-3+粒细胞集落刺激因子刺激的CFU-GM增殖增强无影响。磷脂酰肌醇-3激酶抑制剂LY294002和渥曼青霉素在与白细胞介素-3联合时可替代粒细胞集落刺激因子，因为在LY294002/渥曼青霉素+白细胞介素-3存在下的增殖增强程度与在粒细胞集落刺激因子+白细胞介素-3存在下相同。相反，LY294002和渥曼青霉素在促红细胞生成素存在以及促红细胞生成素+白细胞介素-3存在时抑制增殖。
1）白细胞介素-3与粒细胞集落刺激因子联合以及与促红细胞生成素联合时可能激活不同的信号转导途径；2）不同的信号转导中间体调节红系和髓系祖细胞增殖；3）抑制磷脂酰肌醇-3激酶途径可抑制髓系祖细胞分化，从而增加增殖。
