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，这两种细胞系一直保持在无蛋白培养中。在无血清甲基纤维素培养中，rh EPO、rmIL-3、rh IL-6、rmLIF或rmGM-CSF对L8057Y5细胞集落生长的促进作用大多呈剂量依赖性，这些细胞因子对L8057细胞的集落生长也有促进作用，但幅度较小。EPO-R、IL-6R、gp130、IL-3R亚单位的mRNAs在两株细胞中均有结构性表达。我们还检测了1994年新克隆的rmTpo对骨髓巨核系祖细胞的影响。单用rmTpo刺激巨核细胞集落生长。RmIL-3与rmTpo联合应用不仅使细胞总数增加，而且增加了大克隆和混合型克隆的出现频率。RhEpo和GM-CSF与rmTpo对集落形成有协同作用。重组人转化生长因子β1对BFU-ES和CFU-GM的集落形成无明显影响，但在rmIL-3、rmTPO或两者共同刺激下，CFU-MKs的生长受到明显抑制，提示在巨核细胞生成过程中，转化生长因子β是一个强有力的负调控因子。RT-PCR结果显示Mpl-mRNA在L8057细胞中有表达。这些结果提示，L8057和L8057Y5细胞系对细胞因子的生物学反应具有巨核细胞的特征，这些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。研究巨核细胞中包括TPO在内的各种细胞因子的信号级联反应是全面阐明血小板生成调控机制的有效途径。我们的发现再次验证了L8057细胞对这些目的的有用性。

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.