酪氨酰tRNA合成酶突变体(Y341A)促血小板新生的作用机制研究

Thrombocytopenia is a common side effect for cancer patients treated with chemo or radiotherapy results in a relative decrease in platelets in the blood. A large number of pleiotropic hematopoietic growth factors have been identified to influence various phases of development in megakaryocytic lineage, including recombinant granulocyte-macrophage colony-stimulating factor, stem cell factor, interleukin 1(IL-1), IL-3, IL-6, IL-11, and thrombopoietin (TPO). Although administration of these cytokines reduces the need for platelet transfusions in patients with severe thrombocytopenia，the pleiotropic effect often results in unwanted or unacceptable toxic effects, including hyperbilirubinemia, rapid induction of anemia, fever, fatigue, hypotension, chills, and headache..A lot of researches indicate that many aminoacyl tRNA synthetase possess cytokine function. Latest research reported that human tyrosyl-tRNA synthetase engineered a single tyrosine to alanine amino acid substitution at position 341 unmasked the ELR motif, which made the human tyrosyl-tRNA synthetase (Y341A rhTyrRS(Y341A)) display a kind of thrombopoietic activity. We also found that rhTyrRS(Y341A) helped relieve development of thrombocytopenia in cyclophosphamide (CTX) treated mice; rhTyrRS(Y341A) promoted the migration of megakaryocyte through transwell; And, in transgenic zebra-fish, the number of megakaryocyte in peripheral blood increased when they were treated with rhTyrRS(Y341A). .Different form TPO, rhTyrRS(Y341A) might accelerate the migration and adhesion of megakaryocyte, result in thrombocytopoiesis. However, the Pharmacodynamical mechanism of rhTyrRS(Y341A) is not clear yet..Here, we try to evaluate the protective effect of rhTyrRS(Y341A) against thrombocytopenia induced by chemotherapy and radiation treatment. This research would be carried out through four parts: 1. pharmacodynamics evaluation of rhTyrRS(Y341A); 2. cytokine function research of rhTyrRS(Y341A); 3. mechanism of signal pathway of rhTyrRS(Y341A); 4. The non-TPO thrombopoietic effect of rhTyrRS(Y341A).

最新研究显示酪氨酰tRNA合成酶Y341A突变体(TyrRS(Y341A))可以促血小板新生，与TPO起效方式不同，但其作用机制尚不明确。前期工作制备了TyrRS(Y341A)；证实rhTyrRS(Y341A)预处理给药可缓解化疗诱导的血小板减少；transwell实验显示rhTyrRS(Y341A)对巨核细胞具有趋化作用；转基因斑马鱼用药后，外周血中巨核细胞数目增加。推测TyrRS(Y341A)可能通过NF-κB通路促进内皮细胞VCAM-1表达，趋化巨核细胞迁移黏附，使血小板生成。本项目旨在阐明TyrRS(Y341A)促血小板新生的机制。1.建立动物模型，用TyrRS(Y341A)治疗，判定作用方式。2.利用细胞迁移和黏附实验，研究趋化因子样作用；利用转基因鱼观察细胞迁移和黏附。3.在分子水平，研究促进VCAM-1表达的信号通路。4.利用基因敲除小鼠，证明其非TPO途径的作用。

血小板减少症常见于中晚期恶性肿瘤的放疗、化疗、骨髓移植术后、慢性肝脏疾病以及肝素诱导的免疫性血小板减少性紫癜等疾病。临床上主要治疗手段有血小板输注法。但是血小板输注可引起同种异体免疫反应、输血感染、过敏反应、输血相关的急性肺损伤等，这限制了它们在临床上的使用。因此，需要探索一种更安全的策略来克服和弥补传统策略的不足。研究表明，tRNA 合成酶不仅具有催化 tRNA 氨基酰化的作用，而且具有与细胞因子类似的功能。本研究旨在探讨酪氨酰 tRNA 合成酶Y341A突变体 TyrRS (Y341A) 治疗血小板减少症的疗效及作用机制。通过构建放化疗模型，检测rhTyrRS (Y341A) 对放化疗引起的血小板减少症的疗效。分析 rhTyrRS (Y341A) 对HUVECs 细胞表面 VCAM-1 表达的影响，检测 VEGF-RⅡ/NF-κB 通路的活性，探讨 rhTyrRS (Y341A) 调控VCAM-1 表达的分子机制。研究结果表明，rhTyrRS (Y341A) 可以缓解放化疗引起的血小板减少。RhTyrRS (Y341A) 作用于 HUVEC 细胞膜上的 VEGF-R II 受体。信号通过VEGF-R II 受体由胞外转移到胞内，下游信号通路 NF-κB 发生核易位, 从而导致VCAM-1 启动子的激活。VCAM-1 从细胞内转移到细胞表面，招募更多的巨核细胞粘附在血管壁表面，从而产生血小板。这提示 rhTyrRS (Y341A) 可能是一种安全有效的促血小板生成药物，为放化疗引起的血小板减少症提供新的治疗策略。