Franks Douglas J, Mroske Cameron, Laneuville Odette
Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of Ottawa. 451 Smyth Road, Ottawa, K1H 8M5. Canada. ext. 8330; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa. Canada.
Biol Proced Online. 2001 Dec 12;3:54-63. doi: 10.1251/bpo23.
In platelets, PGHS-1-dependant formation of thromboxane A(2) is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are a-nucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01 which can be induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression. Using a specific antibody to PGHS-1 we have developed a technique utilizing immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein levels as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 x 10(-8) M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen, CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation between PGHS-1 levels and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein level, which shows a consistent increase over the entire course of differentiation, can be used as an additional and better index by which to monitor megakaryocyte differentiation.
在血小板中，由前列腺素内过氧化物合酶-1（PGHS-1）依赖性形成的血栓素A2是血小板功能的重要调节因子，也是阿司匹林对血小板功能进行药理学抑制的靶点。由于血小板是无核细胞，我们使用了永生化的人巨核母细胞系MEG-01，该细胞系在添加佛波酯（TPA）后可被诱导分化为血小板样结构，以此作为研究PGHS-1基因表达的模型系统。我们使用针对PGHS-1的特异性抗体，开发了一种利用免疫荧光显微镜和多个数字图像分析的技术，以监测在8天时间内单次添加TPA（1.6×10⁻⁸ M）诱导MEG-01细胞分化过程中PGHS-1蛋白水平。该方法是流式细胞术的一种快速且经济的替代方法。使用该技术我们观察到TPA诱导了MEG-01细胞的黏附，并且只有未黏附的TPA刺激细胞显示出活力受损。通过血小板特异性细胞表面抗原CD-41的表达来评估MEG-01细胞的分化。后者在MEG-01细胞分化的后期阶段表达。我们证明了PGHS-1水平与MEG-01细胞的整体细胞分化水平之间具有良好的相关性。此外，PGHS-1蛋白水平在整个分化过程中持续增加，可作为监测巨核细胞分化的另一个更好的指标。
