骨髓血管微环境在MLL-AF9急性髓系白血病发病中的作用

Role of Vascular Niche of Bone Marrow in the Development of MLL-AF9 Induced Acute Myclaid Leukemia

目的:探讨骨髓血管微环境在MLL-AF9急性髓系白血病发病过程中的作用。方法:应用移植实验构建未照射MLL-AF9急性髓系白血病模型;利用half-bone免疫染色方法和双光子共聚焦多层多通道多视野成像技术,获取骨髓血管、骨内膜和巨核细胞的图像数据;应用流式细胞术分析不同解剖部位白血病细胞的特性。结果:在MLL-AF9急性髓系白血病发病早期阶段,白血病细胞在股骨干骺端的比例显著高于骨干处;进一步检测凋亡、增殖特性发现,股骨干骺端处的白血病细胞凋亡比例明显低于骨干区,且细胞增殖(S/G2/M期)也明显比骨干区活跃,提示不同解剖部位处白血病细胞的这些特性可能与不同的骨髓微环境有关。3种主要骨髓微环境成分(血管内皮细胞、骨内膜、巨核细胞)的图像数据显示:股骨干骺端和骨干处的血管分布不同;进一步比较白血病细胞与血管内皮细胞、骨内膜、巨核细胞的空间距离结果表明,白血病细胞与血管的空间距离更近,提示血管在白血病发病早期阶段中有重要作用。葡萄糖摄取实验和细胞内ROS检测表明,血管的作用与营养代谢途径无相关性。结论:血管微环境在白血病的发病中起了重要作用,但这种作用不是通过运输营养物质和清除代谢废物途径,可能与血管周的细胞因子或血管的其它作用有关。

Objective:To investigate the role of bone marrow vascular niche in the development of MLL-AF9 acute myeloid leukemia(AML).Methods:Transplantation experiments were performed to establish non-radiated MLL-AF9 AML model;the half-bone immunofluorescence staining and tile-scan imaging of two-photon confocal microscopy were used to obtain the data of 3 main bone marrow niche cells;flow cytometry analysis was performed to characterize leukemia cells in different anatomical sites.Results:In the early stage of MLL-AF9 AML,the proportion of leukemia cells in the metaphysis of the femur was significantly higher than that in the diaphysis.The detection of apoptosis and proliferation rate of leukemia cells showed that the percentage of leukemia cells in metaphysis significantly decreased,and the proliferation(S/G2/M phase)was also significantly more active.These different features of leukemia cells may relate with different bone marrow microenvironment.The image data of 3 major components of bone marrow niche(endothelial cells,endosteum,megakaryocytes)showed different distribution of blood vessels in metaphysis and diaphysis.Furtherly comparing the spatial distance between leukemia cells and endothelial cells,endosteum,megakaryocytes indicated that leukemia cells are closer to the blood vessels,suggesting the important role of blood vessels in the development of leukemia.Glucose uptake assays and intracellular ROS detection showed that the supportive role of blood vessels for leukemia cells did not related with nutrient metabolism pathway.Conclusion:The vascular niche plays an important role in the development of leukemia,and does not relate with the transport of nutrients and the elimination of metabolic waste,instead,which may relate with perivascular cytokines or other vascular functions.