UVRAG介导的自噬在电离辐射引起成熟巨核细胞终末分化障碍中的作用及机制研究

Mature megakaryocytes that lack of proliferation ability were not considered sensitive to ionizing radiation previously. We found that the platelet production of mature megakaryocytes was decreased significantly after 6Gy irradiation. However, its mechanism is unclear. The previous study showed that, after irradiation, the expression and phosphorylation of AKT were reduced, the phosphorylation of ERK1/2 and the expression of LC3II/I were increased, but the phosphorylation of mTOR was not reduced obviously. It was suggested that ionizing radiation may promote autophagy by mTOR independent pathway. We presumed that irradiation may induce ERK1/2 activation and Bcl-2 phosphorylation by down-regulate AKT expression, which promote UVRAG combined to Beclin 1, and lead to autophagy expansion, thus impeding terminal differentiation of mature megakaryocytes and aggravating platelet production disorder. This project aims to analyze changes of ERK1/2, AKT, Bcl-2, UVRAG, autophagy, terminal differentiation and mouse platelet count by RNA interference, western blot, laser confocal microscopy, flow cytometry, electron microscopy and other techniques, aims to investigate the role and mechanism of UVRAG mediated autophagy in mature megakaryocyte terminal differentiation disorder induced by ionizing radiation.

既往认为增殖能力较弱的成熟巨核细胞对电离辐射不敏感。我们发现，经6Gy照射后成熟巨核细胞产板能力显著降低，但机制尚不明确。前期研究发现，照射后成熟巨核细胞中AKT表达及磷酸化均显著降低，ERK1/2磷酸化及LC3II/I增高，但mTOR磷酸化并未显著降低，提示电离辐射可能通过非mTOR依赖途径促进自噬。推测电离辐射可能通过下调AKT表达水平，导致ERK1/2激活，进而通过Bcl-2磷酸化增强了UVRAG与Beclin 1的结并导致自噬扩大，从而阻碍了成熟巨核细胞终末分化并加重了血小板生成障碍。本项目拟采用RNA干扰、免疫共沉淀、Western blot、激光共聚焦、流式细胞分析、电子显微镜观察等技术，分析电离辐射后成熟巨核细胞中ERK1/2、AKT、Bcl-2、UVRAG、自噬、终末分化和小鼠血小板计数的改变，探讨UVRAG介导的自噬在电离辐射引起成熟巨核细胞终末分化障碍中的作用及机制。

既往研究认为增殖能力较弱的晚期成熟巨核细胞对电离辐射不敏感，但我们的研究显示，培养至13天的人脐血CD34+来源原代巨核细胞经6Gy照射后产板能力显著降低，提示电离辐射可能严重影响了成熟巨核细胞的终末分化过程，并进一步加重了辐射损伤后血小板减少症及其并发症的发生，但机制尚缺乏研究。本课题从紫外线抗性相关基因（UVRAG）编码蛋白UVRAG与自噬的联系入手，观察了电离辐射对成熟巨核细胞中UVRAG表达及自噬水平的影响，探讨了电离辐射通过自噬导致成熟巨核细胞产板能力降低的分子机制。课题结论如下：电离辐射通过下调成熟巨核细胞中AKT的表达水平导致ERK1/2通路过度激活，进而增强了UVRAG与Beclin 1结合形成自噬复合体的能力并导致自噬扩大，从而阻碍了成熟巨核细胞终末分化的过程，加重了血小板生成障碍。转染AKT可部分恢复电离辐射引起的成熟巨核细胞ERK1/2磷酸化及UVRAG表达水平升高，从而抑制自噬扩大，并部分恢复电离辐射导致的成熟巨核细胞产板能力及血小板活性下降。这些发现进一步阐释了电离辐射引起血小板生成障碍产生的分子机制，也有助于更好的理解巨核细胞终末分化及血小板生成调控的生理过程。