A Study of the mechanisms regulating the polyploidization and oytokinesisin human megakaryocyte

巨核细胞是一种独特的细胞，在细胞的正常生命周期中有规律地形成多倍体DNA。理解巨核细胞多倍体化的真实本质和控制巨核细胞多倍体化的机制一直受到阻碍，部分原因是我们没有一个合适的模型系统来解决这些问题。为了研究这些问题，我们建立了一种新的因子依赖性人白血病细胞系YMP91。YMP91的增殖依赖于c-kit和gp130的信号。在SCF和IL-6/sIL-6R存在下，通过限制性稀释法建立了四个独立的YMP91亚克隆，分别为YMP91- a、-B、-C和-D。YMP91-C细胞自旋涂片细胞大小不均，10 - 20%可见大多倍体细胞，而YMP91-A细胞大小均匀，未见多核细胞。电镜分析显示，YMP91-A的血小板过氧化物酶活性为阴性，而YMP91-C的血小板过氧化物酶活性为阳性，胞质细结构丰富，提示这些亚系在巨核细胞成熟过程中表现出不同的表型。因此，YMP91及其子基因可望为阐明巨核形成的调控机制提供一个模型系统。我们假设各亚系巨核细胞成熟表型的差异可能是由基因表达的变化引起的。为了确定这些亚系中基因表达的变化，我们利用CDNA微阵列系统分析了超过2304个基因的表达。比较YMP91-A和- C，有53个基因表达水平有显著差异，YMP91-A和- d有23个基因表达水平有显著差异。这些可能在巨核细胞生成中发挥重要作用的候选细胞正在用Northern blot分析进行定量评估，其中一些也正在进行功能评估。

Megakaryocytes are unique cells, developing a polyploid DNA content regularly during the normal life cycle of the cell. Understanding the true nature of megakaryocytic polyploidization and the mechanisms that control polyploidization in megakaryocytes have been hampered partly because we have not had a suitable model system for resolving these issues. To investigate these issues, we have established a novel factor-dependent human leukemia cell line YMP91.The proliferation of YMP91 depends on both signals from c-kit and gp130.Four independent subclones of YMP91, designated YMP91-A, -B, -C, and -D have been established by limiting dilution method in the presence of SCF and IL-6/sIL-6R. The cells in the cytospin smear of YMP91-C are heterogeneous in size, and large polyploid cells can be observed in 10 - 20 %, whereas the cells of YMP91-A are homogeneous in size and there are no multinucleated cells. Platelet peroxidase activity examined by electron microscopic analysis of YMP91-A is negative, but that of YMP91-C is positive along with abundant cytoplasmic fine structures, suggesting these sublines show distinct phenotypes in respect of megakaryocytic maturation. YMP91 and its sublines may thus be expected to serve a model system for clarifying the mechanisms regulating the megakaryopoiesis. We have hypothesized that the difference of phenotypes in terms of megakaryocytic maturation in each subline should be caused from the changes in gene expression. To identify the changes in gene expression in these sublines, we have utilized a CDNA microarray system to analyze the expression of over 2,304 genes. Comparison of YMP91-A and - C revealed 53 genes that showed significant difference in the expression level, and that of YMP91-A and -D did 23 genes. These candidates that might play an important role in megakaryocytopoiesis are under investigation with Northern blot analysis for quantitative evaluation and some of them are also being functionally evaluated.