EPOR在红系终末发育阶段的功能及分子机制研究

There are two different erythropoiesis waves according to the tissue and time: primitive erythropoiesis and definitive erythropoiesis. Both of them include early-stage erythropoiesis, terminal erythropoiesis differentiation and reticulocyte maturation. Erythropoiesis is a complicated and precisely regulated biological process, of which EPO/EPOR pathway plays an essential role. Previous studies demonstrated that EPOR-Null mice would die in E13.5 with a block in definitive erythropoiesis at the late progenitor (CFU-E) stage but slight defect in primitive erythropoiesis. Thus, we suppose that EPO/EPOR plays different roles in primitive and definitive erythropoiesis. Meanwhile, due to the block in early-stage erythropoiesis, the function of EPOR in terminal erythropoiesis differentiation is unclear. To reveal the molecular mechanism of EPO/EPOR in terminal differentiation erythropoiesis, we have constructed the Gypa-tdTomato-iCre and EPOR-Loxp mouse models by which we may knockout the EPOR in proerythroblast to focus on the different functions of EPOR in terminal differentiation of primitive and definitive erythropoiesis. This proposal we plan to elucidate the regulatory function of EPOR in different-stage erythroblasts by flow cytometry, transcriptomics and mouse model. Our work will reveal the biological function of EPOR in erythropoiesis and may shed light on the understanding of disrupted erythropoiesis.

红细胞发育根据位置和时间可以分为两波：原始红系造血和终末红系造血。这两波均可分为红系发育早期、红系终末发育和网织红细胞成熟三个阶段。红细胞发育是一个复杂而精确的过程，其中EPOR通路发挥了最基本的调控功能。以往利用EPOR敲除小鼠的研究表明终末红系造血被阻滞在CFU-E阶段（红系发育早期）而导致小鼠在13.5天死亡，原始红系造血虽受到影响但相对较小。由此推测，EPOR在原始红系造血和终末红系造血会有功能差异。由于红细胞被阻滞在早期阶段，因此对于EPOR在终末发育阶段的功能并不清楚。为进一步研究EPOR在红系终末发育阶段的功能，利用已经构建好的Gypa-tdTomato-iCre和EPOR-loxp小鼠，特异性地在红系终末阶段敲除EPOR，利用流式、转录组学、动物实验等手段研究两波造血中红系终末发育阶段EPOR的不同功能，从而完善EPOR在造血系统中的生物功能，为阐明红系发育紊乱奠定基础。

红细胞发育根据位置和时间可以分为两波：原始红系造血和终末红系造血。这两波均可分为红系发育早期、红系终末发育和网织红细胞成熟三个阶段。红细胞发育是一个复杂而精确的过程，其中EPOR通路发挥了最基本的调控功能。以往利用EPOR敲除小鼠的研究表明终末红系造血被阻滞在CFU-E阶段（红系发育早期）而导致小鼠在13.5天死亡，原始红系造血虽受到影响但相对较小。由此推测，EPOR在原始红系造血和终末红系造血会有功能差异。由于红细胞被阻滞在早期阶段，因此对于EPOR在终末发育阶段的功能并不清楚。在此研究中，我们构建了Gypa-tdTomato-iCre和EPOR-loxp小鼠，特异性地在红系终末阶段敲除EPOR。首先我们分析了原始红系造血中EPOR，通过分离小鼠胚胎E9.5，E10.5，E11.5和E12.5天的外周血细胞中primitive proerythroblasts，basophilic erythroblasts，polychromatophilic erythroblasts和orthochromatic erythroblasts，并通过测序进一步分析了这些原始红系造血不同阶段细胞的转录组图谱，明确EPOR在原始红系造血中的表达。综上所述，本项目首次绘制了原始红系造血不同阶段的基因表达情况。项目的完成拓展了我们对小鼠原始红系造血的认识，完善了EPOR在原始红系造血和终末红系造血中的功能。