Broudy V C, Lin N L, Kaushansky K
University of Washington, Division of Hematology, Seattle, 98195, USA.
Blood. 1995 Apr 1;85(7):1719-26.
Thrombopoietin (Tpo), the ligand for the c-mpl receptor, is a major regulator of platelet production in vivo. Treatment of mice with purified recombinant Tpo increases platelet count fourfold and expands colony-forming unit-megakaryocyte (CFU-Meg) numbers. Other cytokines including interleukin-3 (IL-3), IL-6, IL-11, erythropoietin (Epo), and stem cell factor (SCF) can stimulate megakaryopoiesis. Therefore, we examined the effects of recombinant murine Tpo in combination with these cytokines on megakaryopoiesis in vitro. Murine marrow cells were cultured in agar in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% horse serum and beta-mercaptoethanol in the presence of recombinant growth factors, and CFU-Meg colonies were counted on day 5. Megakaryocyte ploidy was analyzed using murine marrow cells cultured for 5 days in IMDM supplemented with 1% nutridoma-SP and recombinant growth factors. Megakaryocytes were identified by labeling with the 4A5 antibody and ploidy was analyzed by flow cytometry. Tpo supported the growth of CFU-Meg in a dose-dependent manner. Although the addition of SCF (50 ng/mL), Epo (2 U/mL), or IL-11 (50 ng/mL) alone exerted only a modest effect on CFU-Meg growth, the combination of SCF plus Tpo, Epo plus Tpo, or IL-11 plus Tpo resulted in a synergistic enhancement of the number of CFU-Meg colonies. IL-3 alone supported CFU-Meg colony growth, and the effects of IL-3 plus Tpo or IL-6 plus Tpo on colony growth appeared to be approximately additive. Fifty percent of megakaryocytes generated in cultures containing IL-3 or Epo displayed < or = 16 N ploidy. In contrast, cultures containing Tpo uniquely generated large numbers (30% to 35% of the total) of megakaryocytes with > or = 64N ploidy. These results show that Tpo stimulates both proliferation of committed megakaryocytic progenitor cells and maturation of megakaryocytes, and that two multipotent cytokines, SCF and IL-11, as well as a late-acting erythroid cytokine, Epo, can synergize with Tpo to stimulate proliferation of CFU-Meg.
血小板生成素（Tpo）是c-mpl受体的配体，是体内血小板生成的主要调节因子。用纯化的重组Tpo治疗小鼠可使血小板计数增加四倍，并增加集落形成单位-巨核细胞（CFU-Meg）数量。包括白细胞介素-3（IL-3）、IL-6、IL-11、促红细胞生成素（Epo）和干细胞因子（SCF）在内的其他细胞因子可刺激巨核细胞生成。因此，我们研究了重组鼠Tpo与这些细胞因子联合对体外巨核细胞生成的影响。将鼠骨髓细胞在含有10%马血清和β-巯基乙醇的伊斯科夫改良杜尔贝科培养基（IMDM）中，于重组生长因子存在的情况下在琼脂中培养，在第5天计数CFU-Meg集落。使用在补充有1%营养瘤-SP和重组生长因子的IMDM中培养5天的鼠骨髓细胞分析巨核细胞倍性。通过用4A5抗体标记鉴定巨核细胞，并通过流式细胞术分析倍性。Tpo以剂量依赖性方式支持CFU-Meg的生长。虽然单独添加SCF（50 ng/mL）、Epo（2 U/mL）或IL-11（50 ng/mL）对CFU-Meg生长仅产生适度影响，但SCF加Tpo、Epo加Tpo或IL-11加Tpo的组合导致CFU-Meg集落数量的协同增强。单独的IL-3支持CFU-Meg集落生长，IL-3加Tpo或IL-6加Tpo对集落生长的影响似乎大致呈相加作用。在含有IL-3或Epo的培养物中产生的巨核细胞中有50%显示倍性≤16N。相比之下，含有Tpo的培养物独特地产生大量（占总数的30%至35%）倍性≥64N的巨核细胞。这些结果表明，Tpo刺激定向巨核细胞祖细胞的增殖和巨核细胞的成熟，并且两种多能细胞因子SCF和IL-11以及一种晚期作用的红细胞生成细胞因子Epo可与Tpo协同刺激CFU-Meg的增殖。
