利用斑马鱼cebpα突变体分析髓系造血调控机制并建立白血病疾病模型的研究

Myeloid cells are central cellular components of blood system and essential regulators for the host defense, embryo/organo-genesis, and tissue regeneration. In vertebrates, myeloid cells are classified into two major lineages: granulocytic and monocytic lineages. Granulocytic lineage consists of neutrophils, eosinophils, basophils, and mast cells; and they are the key effectors of generating innate immunity. On the other hand, monocytic lineage, which includes the circulating monocytes and tissue-resident macrophages, plays crucial roles not only in inflammatory response but also actively participates in embryo/organo-genesis, tissue homeostasis, and tissue regeneration. The development of myeloid cells is tightly controlled and irregularities in their development are closely associated with the onset and progression of a variety of human disorders including myeloid leukemia. .Studies have shown that myeloid cell development is predominantly governed by transcription factors, and mutations in those key regulators are attributed to the occurrence of myeloid leukemia in human patients. With its several unique advantages, zebrafish have emerged as an excellent model organism for the study of vertebrate development and human disorders and for drug screening. We recently isolated a myeloid-defective zebrafish mutant, cebpαmus6 which harbors a loss-of-function mutation in Cebpα, a well-known transcription factor essential for myelopoiesis in mammals. ~15% of human acute myeloid leukemia (AML) patients are caused by the decrease or loss of C/EBP? function. Preliminary characterization showed that myelopoiesis in cebpαmus6 mutants was severely impaired as indicated by the absence of granulocytic and monocytic lineages. Our preliminary result indicates that Cebpα plays a conserved biological function from teleost fish to human. .Here we propose to dissect the role of Cebpα in myeloid cell development and to create AML zebrafish models by genetically modifying cebpαmus6 mutant fish. We also like to explore the co-operative effect between Cebpα and two other myeloid regulators Pu.1 and Runx1 in the onset and progress of AML. Our proposed study will contribute to our understanding of the genetic networks underlying myeloid development and establish zebrafish AML models for drug discovery.

转录因子Cebpα在鼠胎儿及成年髓系造血中发挥着重要作用，其功能下降或缺失与人类髓系急性白血病密切相关。.我们的前期工作获得一个斑马鱼突变体，其早期髓系造血被阻断，导致髓系细胞（巨细胞系和粒细胞系）的缺失。基因定位显示突变基因为cebpα，突变体命名为cebpαsmu6。.本项目拟分析Cebpα在髓系造血中的作用并通过基因修饰cebpαsmu6突变体成鱼建立急性髓系白血病（AML）斑马鱼疾病模型，从而进一步研究Cebpα与另外两个已知髓系转录因子Pu.1和Runx1之间的相互关系，以便解释髓系发育和急性髓系白血的基因调控网络并可望提供一个治疗AML药物筛选的疾病模型。

已知转录因子Cebpα在髓系造血中发挥重要作用，其功能下降或缺失与人类髓系造血系统疾病（如白血病）关系密切。斑马鱼是研究髓系造血系统疾病较合适的动物模型。为了进一步研究Cebpα及其相关基因突变对造血功能的影响及机制，我们在前期通过ENU（N-ethyl-N-nitrosourea）化学诱变大规模正向遗传学突变筛选的方法获得了斑马鱼cebpαsum6-/- （此后更名为molihkz7）突变体。本项目通过测序分析发现，突变体在cebpα存在插入性的功能缺失突变位点。通过运用活体成像追踪及细胞死亡及增殖分析等实验，我们发现Cebpα的缺陷导致胚胎髓系前体细胞停留在细胞分裂的S期，导致前体细胞的维持及分化缺陷。已知Runx1对髓系细胞分化成单核/巨噬细胞和粒细胞中发挥重要作用。为了研究Cebpα和Runx1在髓系细胞发育过程中的相互作用，我们构建了能使巨噬细胞数量增多，而粒细胞数量减少的runx1w84x-/-突变体。将cebpαsum6-/- （即molihkz7）突变体和runx1w84x-/-突变体杂交，发现Cebpα和Runx1可以协同调控胚胎粒细胞的发育，对巨噬细胞发育影响不明显。这些数据说明Cebpα能通过调控胚胎髓系细胞前体细胞的正常增殖来维持髓系粒细胞的正常发育。此外，我们还构建了pu.1G242D斑马鱼突变体，阐明了pu.1G242D突变的斑马鱼髓系细胞增生异常，表明pu.1在维持骨髓前体细胞正常分化和增殖的过程中是必须的，也提示pu.1在抑制骨髓增生异常中的重要作用。本项目探讨了Cebpα、Pu.1、Runx1等对早期胚胎髓系细胞发育的调控机制，进一步明确了这些基因对造血过程的影响。通过本项目的研究，我们构建了一批影响斑马鱼髓系造血的突变体，如：pu.1G242D斑马鱼突变体、cebpαsum2-/-、cebpαsum3-/-和cebpαsum3-/-、runx1w84x-/-。这些突变体模型为研究髓系造血相关疾病（如白血病）的治疗或者药物靶向提供了新的工具。