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

巨核细胞是独特的细胞，在细胞的正常生命周期中有规律地形成多倍体DNA含量。了解巨核细胞多倍体化的真正本质和控制巨核细胞多倍体化的机制一直受到阻碍，部分原因是我们还没有一个合适的模型系统来解决这些问题。本研究建立了一种新的因子依赖性人白血病细胞系YMP 91。YMP 91的增殖依赖于c-kit和gp 130的双重信号。在SCF和IL-6/sIL-6 R存在下，采用有限稀释法建立了4个独立的YMP 91亚克隆，命名为YMP 91-A、-B、-C和-D。YMP 91-C细胞离心涂片中的细胞大小不均匀，在10 - 20%中可以观察到大的多倍体细胞，而YMP 91-A细胞大小均匀，没有多核细胞。电镜分析表明，YMP 91-A的血小板过氧化物酶活性为阴性，而YMP 91-C的血小板过氧化物酶活性为阳性，沿着伴有丰富的细胞质精细结构，表明这两个亚系在巨核细胞成熟方面表现出不同的表型。因此，YMP 91及其亚系有望成为阐明巨核细胞发生调控机制的模型系统。我们推测，各亚系巨核细胞成熟的表型差异是由基因表达的变化引起的。为了鉴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2，304个基因的表达。比较YMP 91-A和- C的表达量，发现有53个基因表达量有显著差异，而YMP 91-A和-D的表达量有23个基因差异。这些可能在巨核细胞生成中发挥重要作用的候选者正在用北方印迹分析进行定量评价，其中一些也正在进行功能评价。

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.