Genetic Analysis of Megakaryopoiesis and Its Diagnostic Application

我们目前的研究旨在阐明为什么相同的巨核细胞祖细胞在不同的巨核细胞发育阶段对不同的细胞因子有反应。首先，我们分析了白细胞介素3受体亚基（IL-3R） mRNA和IL-6受体（IL-6R） mRNA在核细胞分化过程中的表达。细胞因子与其受体结合产生多种生物学反应的分子机制尚不清楚。为了阐明巨核细胞分化过程中的细胞内信号通路，我们研究了ras p21样小GT p结合蛋白（如smg p21和rho p21）在巨核细胞发育中的作用。此外，我们试图建立具有巨核细胞谱系的细胞系，这些细胞系对白血病抑制因子（LIF）、IL-3、GM-CSF和11-6等细胞因子无反应。我们对该细胞系进行了表征，以阐明巨核细胞白血病细胞增殖和分化的机制。我们的结果如下：我们发现il - 3r mRNA在CMK细胞中呈组成性表达，并被TPA和IL-6（巨核细胞分化剂）短暂下调，而IL-6 mRNA的表达在TPA的作用下增加，并与巨核细胞的分化有关。这些结果表明，细胞因子受体mRNA的表达调控可能是巨核细胞发育过程中巨核祖细胞对不同细胞因子反应的调控机制之一。在CMK细胞中检测到smg p21 mRNA和smg P21B mRNA， TPA显著提高了smg p21 mRNA和smg P21B mRNA的表达水平，使CMK细胞向更成熟的巨核细胞分化。在tpa诱导的CMK细胞分化过程中，smg - p21蛋白分子也增加。TPA使糖蛋白（GP）IIb mRNA水平升高，但smg p21 mRNA水平升高的时间过程比GP IIb mRNA水平升高的时间过程要快。这些结果提示smgp21可能在巨核细胞分化调控中起重要作用。CMK细胞组成性地表达rho A和rho C mrna。此外，C3外泌酶（C3）的加入导致CMK细胞中rho蛋白失活，从而导致巨核细胞多倍体化。这一结果表明rho蛋白可能是巨核细胞多倍体化的调节因子。新的巨核细胞白血病细胞系，命名为KHI84，是从患者身上建立的，该患者最初的症状是右侧颈部淋巴结肿胀。表面标记分析显示KHI84细胞表达GP Ib和GP IIb/IIIa抗原。然而，Northern blot分析显示，KHI84细胞中缺乏成熟巨核细胞标志物血小板因子4 mRNA。此外，LIF、IL-3、GM-CSF、IL-6等细胞因子和TPA均未引起KHI84细胞的增殖和/或分化。这些发现表明KHI85是研究巨核生成机制的有用细胞系。少

Our present study was designed to clarify why the same megakaryocyte progenitor cells respond to various cytokines at different stages of magakaryocyte development. First, we analyzed the expressions of IL-3 receptor bata-subunit(IL-3R) mRNA and IL-6 receptor(IL-6R) mRNA during magakaryocyte differentiation. The molecular mechanism by which cytokine binding to its receptor exerts diverse biological responses remains unknown. To clarify the intracellular signal pathway in megakartocyte differentiation, we examined the role of ras p21 -like small GT P-binding proteins such as smg P21 and rho P21 in megakaryocyte development. In addition, we tried to establish the cell line with megakaryocyte lineage, which are unresponsive to cytokines such as leukemia inhibitory factor(LIF), IL-3, GM-CSF, and 11-6. We characterized that cell line to eluciade the mechanism by which the megakaryocytic leukemia cell proliferates and differentiates. Our results are as follows.1. We found that IL-3 R mRNA wa … More s expressed constitutively in CMK cells and was transiently down-regulated by TPA and IL-6(megalaryocyte-differentiating agents), while the expression of IL-6 mRNA was increased by TPA in association with the differentiation of megakaryocytes. These results provides evidence that regulation of expression of cytokine receptor mRNA may be one the mechanisms for modulating the responsiveness of megakaryocyte progenitor cells to different cytokines during megakaryocyte development.2. Both smg p21 mRNA and smg P21B mRNA were detected in CMK cells, and their levels were markedly elevated by TPA, which caused the differentiation of CMK cells into more mature megakaryocytes. The smg p21 protein molecules also increased during the TPA-induced diffrentiation of CMK cells. The mRNA level of glycoportein(GP)IIb was increased by TPA, but the time course of increase in the smg p21 mRNA levels was more rapid than that of the increase in the GP IIb mRNA level. These results imply that smg P21 may play an important role in regulating the megakaryocyte differentiation.3. CMK cells constitutively expressed rho A and rho C mRNAs. Further, addition of C3 exoenzyme(C3) caused the inacitivation of rho protein in CMK cells and thereafter the megakaryocyte polyploidization. This result reveals that rho proteins may function as a regulator of the megakaryocyte polyploidization.4. The new megakaryocytic leukemia cell line, designated KHI84, was established from the patient, who had the initial symptoms of swelling of the right cervical lymphnode. Surface marker analysis showed that KHI84 cells expressed GP Ib and GP IIb/IIIa antigens. However, Northern blot analysis revealed that platelet factor 4 mRNA, a mature megakaryocyte marker, was absent in KHI84 cells. In addition, neither cytokines such as LIF, IL-3, GM-CSF, and IL-6 nor TPA caused the proliferation and/or differentiation of KHI84 cells. These findings indicate that KHI85 is a useful cell line for investigating the mechanism of megakaryopoiesis. Less