Majka M, Ratajczak J, Baj-Krzyworzek M, Kijowski J, Reca R, Machaliński B, Kubicze K, Ratajczak M Z
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Folia Histochem Cytobiol. 2001;39(3):235-44.
The aim of this study was to learn more on the role of chemokines in the regulation of human megakryopoiesis. Normal human megakaryoblasts were expanded in serum-free liquid cultures and subsequently (1) phenotyped for expression of various chemokine receptors, (2) evaluated if chemokine receptors which they express are functional after stimulation by chemokines (calcium flux assay, chemotaxis, phosphorylation of MAPK-p42/44 and AKT proteins), and (3) investigated for expression and secretion of selected chemokines by employing RT-PCR and ELISA assays, respectively. In addition we also phenotyped peripheral blood platelets for expression of chemokine receptors and chemokines. We found that while human megakaryoblasts express several chemokine receptors (CXCR4, CCR6, CCR8, CCR5, CCR2 and CXCR3), CXCR4 was the only receptor detectable by FACS on human platelets. Moreover, among various chemokines tested, only SDF-1 (CXCR4 ligand) stimulated calcium flux and chemotaxis in normal human megakaryoblasts and phosphorylated MAPK-p42/44 and AKT in these cells. Although mRNAs for several chemokines were detectable by RT-PCR in normal human megakaryoblasts, only RANTES, IL-8, MCP-1 and PF-4 were found to be secreted by these cells. Finally we noticed that no chemokine tested in this study affected CFU-Meg colony formation by human CD34+ cells in serum-free cultures. We conclude that from all the chemokine receptor-chemokine axes tested, only SDF-1-CXCR4 axis was functional in assays employed in our studies, which further support the view that this axis plays a privileged role in regulating normal human megakaryopoiesis.
本研究的目的是进一步了解趋化因子在人类巨核细胞生成调控中的作用。正常人类巨核母细胞在无血清液体培养中扩增，随后：（1）对各种趋化因子受体的表达进行表型分析；（2）评估它们所表达的趋化因子受体在趋化因子刺激后是否具有功能（钙流测定、趋化性、MAPK-p42/44和AKT蛋白的磷酸化）；（3）分别采用RT-PCR和ELISA测定法研究选定趋化因子的表达和分泌。此外，我们还对外周血血小板的趋化因子受体和趋化因子表达进行了表型分析。我们发现，虽然人类巨核母细胞表达多种趋化因子受体（CXCR4、CCR6、CCR8、CCR5、CCR2和CXCR3），但CXCR4是人类血小板上唯一可通过流式细胞术检测到的受体。此外，在测试的各种趋化因子中，只有SDF-1（CXCR4配体）能刺激正常人类巨核母细胞中的钙流和趋化性，并使这些细胞中的MAPK-p42/44和AKT磷酸化。虽然通过RT-PCR在正常人类巨核母细胞中可检测到几种趋化因子的mRNA，但这些细胞仅分泌RANTES、IL-8、MCP-1和PF-4。最后，我们注意到本研究中测试的任何趋化因子均不影响无血清培养中人类CD34+细胞的CFU-Meg集落形成。我们得出结论，在我们研究中采用的测定方法中，所有测试的趋化因子受体-趋化因子轴中，只有SDF-1-CXCR4轴具有功能，这进一步支持了该轴在调节正常人类巨核细胞生成中起特殊作用的观点。
