急进高原习服期红细胞miR-144-5p通过外泌体对CD34+造血祖细胞增殖分化的作用机制研究

Erythremia universally occurs in people after rush entry into plateau. Researches show that erythropoietin (EPO) plays a major role on erythremia at the early stage of rapid entering plateau, but the EPO concentration decreases at the stage of high-altitude acclimatization, whereas the number of red blood cells (RBCs) remains at a high level, suggesting that there are other mechanisms for the regulation of erythropoiesis. It has been proved that multiple miRNAs can affect the proliferation and differentiation of CD34+ hematopoietic progenitor cells and then regulate erythropoiesis. We previously found that erythrocyte-derived miR-144-5p was significantly downregulated at the stage of plateau acclimatization and released into plasma by exosomes, and exosomes secreted by erythrocytes promoted the proliferation of CD34+ hematopoietic progenitor cells. We propose that the high-altitude downregulated miRNA may be another important regulator of erythropoiesis in addition to EPO. This subject will make clear the way of “erythrocyte-derived miRNAs→ exosomes→ CD34+ hematopoietic progenitor cells → formation of mature RBCs”by means of fluorescence tracing, flow cytometry and other techniques; by RT-PCR and western blot analysis, the study will verify the effect and mechanism of RBC miR-144-5p on proliferation and differentiation of the CD34+ hematopoietic progenitor cells by regulating the expression levels of target genes. Finally, a scientific basis is provided for improving the regulation mechanism of erythremia in the high-altitude hypoxia environment.

红细胞增多是急进高原人群普遍存在的生理变化。研究表明促红细胞生成素（EPO）对急进高原初期红细胞增多发挥主要作用，但在高原习服期EPO浓度下降红细胞数量依然维持在高水平，提示存在其他的红细胞生成调节机制。已证实miRNA能影响CD34+造血祖细胞增殖分化进而调节红细胞生成。我们前期发现红细胞miR-144-5p在高原习服期显著下调，并通过外泌体释放入血浆；且红细胞外泌体能促进CD34+造血祖细胞的增殖。我们提出红细胞来源的miR-144-5p可能是除EPO外红细胞生成的重要调节因子，本课题拟通过荧光示踪、流式细胞术等明确“红细胞miR-144-5p→外泌体→CD34+造血祖细胞→成熟红细胞生成”的作用途径；通过定量PCR、免疫印迹分析等阐明miR-144-5p通过调节CD34+造血祖细胞中靶基因的表达进而影响其增殖分化的作用机制。本项目为完善高原低氧环境下红细胞增多的调节机制提供科学依据。

急进高原人群中普遍存在红细胞增多等生理变化。已有研究发现促红细胞生成素（EPO）对急进高原初期红细胞增多发挥主要作用，在高原习服期，EPO浓度显著下降但是红细胞的数量依然维持在高水平，这提示可能存在其他的红细胞生成调节机制。已有文献证实miRNA能影响CD34+造血祖细胞增殖分化进而调节红细胞生成。我们此次整个研究的完成，明确了如下的研究内容：通过高通量测序及定量PCR等，我们完成了世居高原人群和平原地区人群红细胞miRNA测序结果对比分析，筛选到差异表达miRNA—miR-144-5p；通过高通量测序及定量PCR等，我们完成了急进高原人群红细胞miRNA的差异表达分析，明确红细胞miR-144-5p是急进高原习服期的效应miRNA；通过percoll密度梯度离心、高通量测序及定量PCR等，我们完成了不同天龄红细胞miRNA的差异表达分析，明确了miR-144-5p会随着红细胞天龄的增长在红细胞中累积而表达升高；通过荧光示踪、定量PCR、免疫印迹分析等，我们完成了红细胞外泌体的荧光定位检测及其携带miR-144-5p对造血祖细胞系的作用分析，明确红细胞可能会随着天龄的增加携带更多的miR-144-5p进入造血祖细胞进而调节其增殖分化。本项目最终的实验结果为完善高原低氧环境下红细胞增多的调节机制提供科学依据。