Studies on regulatory mechanisms of megakaryocytepoiesis using the cell line grown in protein-free culture.

我们检测了细胞因子对小鼠巨核细胞系L8057和L8057Y5生长的影响，它们在无蛋白培养中得到维持。在无血清甲基纤维素培养中，rhEPO、rmIL-3、rhIL-6、rmLIF或rmGM-CSF的加入促进了L8057Y5细胞的集落生长，主要呈剂量依赖性。L8057细胞的集落生长也受到这些细胞因子的促进，但程度较低。EPO-R、IL-6R、gp130、IL-3R亚基mrna在两种细胞系中均有组成性表达。我们还测试了1994年新克隆的rmTpo对骨髓巨核细胞祖细胞的影响。通过刺激脚底rmTpo观察巨核细胞集落生长。rmIL-3与rmTpo的添加不仅增加了总数，而且增加了大菌落和混合型菌落的频率。rhEpo、GM-CSF与rmTpo在菌落形成方面均表现出协同作用。添加rhTGF-beta_1后，BFU-Es和cfu - gm的菌落形成没有显著减少；然而，在rmIL-3、rmTPO或两种细胞因子的刺激下，CFU-Mks的生长明显受到抑制，这表明tgf - β在巨核细胞生成中是一种有效的负调节因子。rmTpo对L8057细胞的生长也有促进作用，主要表现为浓度依赖性。RT-PCR显示Mpl-mRNA在L8057细胞中的组成性表达。这些结果表明，L8057和L8057Y5细胞系对细胞因子的生物学反应具有巨核母细胞的特征，细胞因子对细胞系的促生长作用可能是通过特异性受体实现的。研究巨核母细胞中包括Tpo在内的各种细胞因子的信号级联，是全面阐明血小板形成调控机制的重要途径。我们的发现再次证实了L8057细胞在这些方面的有效性。

We examined the effects of cytokines on the growth of murine megakaryocytic cell lines, L8057 and L8057Y5, which had been maintained in protein-free culture. In serum-free methylcellulose culture, the addition of rhEPO,rmIL-3, rhIL-6, rmLIF,or rmGM-CSF enhanced colony growth of L8057Y5 cells mostly in dose-dependent fashion Also, colony growth of L8057 cells was enhanced by these cytokines, but to a lesser extent. The expression of mRNAs for EPO-R,IL-6R,gp130, IL-3R subunits were constitutively expressed in both lines. We also tested the effect of rmTpo, which was newly cloned in 1994, on the megakaryocyte progenitors in marrow. The megakaryocyte colony growth was observed by the stimulation of sole rmTpo. The addition of rmIL-3 with rmTpo showed increase not only in the total number, but in the frequencies of large colonies and of mixed-type colonies. Also, rhEpo and GM-CSF showed synergistic effects with rmTpo on the colony formation. Colony formation by BFU-Es and CFU-GMs was not significantly reduced by the addition of rhTGF-beta_1 ; however, the growth of CFU-Mks, stimulated by rmIL-3, rmTPO,or both cytokines, was markedly inhibited, which suggest that TGF-beta is a potent negative-regulator in megakaryocytepoiesis.rmTpo also enhanced the growth of L8057 cells mostly in a concentration-dependent fashon as observed in the growth of CFU-Mk. RT-PCR revealed the costitutive expression of Mpl-mRNA in L8057 cells. These results suggest that L8057 and L8057Y5 cell lines have the characteristics of megakaryoblastic cells in their biological responses to cytokines, and that the growth-enhancing effects of these cytokines on the cell lines may be achieved through specific receptors. For totally clarifying the regulation mechanisms of thrombopoiesis, it should be promissing way to investigate signaling cascades for every cytokines including Tpo in megakaryoblastic cells. Again our findings validate the usefulness of L8057 cells for these purposes.