The Analysis of Signal Transduction System in the Activation of Proplatelet Formation of Megakaryocytes

巨核细胞前血小板形成激活信号转导系统分析

• 批准号: 13671075
• 负责人: ISHIDA Yoji
• 金额: $ 2.18万
• 依托单位: Iwate Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671075/
• 关键词: proplatelet formation; platelet production; AT III; HDL; protein kinase C; Rho kinase; prplatelet formation; 巨核球胞体突起形成(PPF); PPF刺激因子; 巨核芽球性白血病細胞株; アンチトロンビンIII; トロンビン

1. Purification of proplatelet formation (PPF) stimulatory factor.We purified the PPF stimulatory factor from the normal human plasma and determined the amino acid sequence of the purified protein. It was antithrombin III, which in vitro activity expressed in association with high density lipoprotein (HDL).2. Selection of human megakaryoblastic cell line, which respond to PPF stimulatory factor.We established the quantitative assay system in which MEG cell line expressed PPF in response to AT III/HDL. AT III/HDL stimulated PPF of MEG cell line in a dose dependent manner (HDL 0 μg/ml 1.1%, HDL 50 μg/ml 30.8%, HDL 100 μg/ml 46.5%, HDL 200 μg/ml 57.8%).3. Inhibition of PPF expression or not in the above assay system with the incubation of kinase inhibitors. PD98059(MAPK inhibitor), LY294002(PI3K inhibitor), SB203580(p38 MAPK inhibitor), KT5720(PKA inhibitor), BIM(PKC activator inhibitor) or Y27632(Rho kinase inhibitor) was added in the above assay system. PPF expression of MEG cell line was inhibited by the addition of BIM or Y27632 (BIM : 100 μM 70.7%, 500 nM 66.9%, 1 μM 54.0%, 10 μM 4.5%, 100 μM 5.6%, Y27632 :100 nM 97.6%, 1 μM 88.5%, 10 μM 61.3%).Based on the results, PPF expression of megakaryocytes was deeply involved in the transduction systems of protein kinase C and Rho kinase.

1.血小板前体形成刺激因子的纯化：从正常人血浆中纯化了血小板前体形成刺激因子，并测定了其氨基酸序列。其体外活性与高密度脂蛋白（HDL）相关.选择对PPF刺激因子有反应的人巨核细胞系，建立了MEG细胞系对AT Ⅲ/HDL反应性PPF表达的定量检测体系。AT Ⅲ/HDL以剂量依赖方式刺激MEG细胞的PPF（HDL 0 μg/ml 1.1%，HDL 50 μg/ml 30.8%，HDL 100 μg/ml 46.5%，HDL 200 μg/ml 57.8%）.在上述测定系统中与激酶抑制剂孵育是否抑制PPF表达。在上述试验系统中加入PD 98059（MAPK抑制剂）、LY 294002（PI 3 K抑制剂）、SB 203580（p38 MAPK抑制剂）、KT 5720（PKA抑制剂）、BIM（PKC激活剂抑制剂）或Y27632（Rho激酶抑制剂）。结果表明，BIM和Y27632对MEG细胞PPF表达的抑制率分别为70.7%，500 nM 66.9%，1 μM 54.0%，10 μM 4.5%，100 μM 5.6%，Y27632：100 nM 97.6%，1 μM 88.5%，10 μM 61.3%。

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