基于Irf8的突变体建立斑马鱼白血病模型

Leukemia is a harmful disease resulted from the defect of leukocytes development or leukopoiesis. Although the mechanism of leukopoiesis and leukemia formation is being exploited continously until now, it has not been well dissected yet, which prevents the progress in the cure of leukemia. Up to now, the best way to treat leukemia is bone marrow transplantation, which is limited by the requirement of the blood type matchment and repression of immunal rejection as well as high cost unfortunately. As a result, chemical drug treatment is still the first choice in clinical. In order to discover more effective chemical drugs and explore the molecular mechanism inside,our lab decided to establish the zebrafish leukemia especially myeloid leukemia model based on the myelopoiesis defect mutant, which will be utilized for the High Throughout Screening (HTS) of the chemical drugs useful for leukemia treatment. With this as the goal, our lab firstly knocked out irf8, a well known factor functioning in the myeloid cell development and myeloid leukemia formation, genetically in zebrafish by TALEN. We successfully obtained the germ-transmition mutant fish. The preliminery data indicated that the irf8 mutant homozygous could survive beyond adult and manifested disturbed myeloid cells type, such as the expand of neutrophils and short of macrophages. This phenotype is quite similar to the irf8 knockout mice which suffer chronic myeloid leukemia. Therefore, irf8 mutant homozygous should be an ideal candidate for the establishment of zebrafish leukemia model. Meanwhile, the pu.1G240D mutant zebrafish in our hand is already proved to be a valuable candidate for the human M2 type AML model. Additionaly, the Tg(coro1a:eGFP;lyz:Dsred) created by us could distinctly label both macrophages and neutrophils, which facilitated the observation of leukocyte situation and made chemical screening more feasible. Taking advantage of these mutants and transgenic fish lines, we will carry out the HTS to find out the valuable chemical drugs in leukopoiesis and leukmemia in this project, and dig out the mechanism subsequently.

白血病是一类极其严重的血液系统疾病，具有较高的致死率，危害极大。白血病的发生发展是白细胞异常发育的结果，因此研究白细胞的发育机理并寻找能够治疗白血病的新型小分子药物具有极大的社会意义。本项目通过TALEN技术构建了白细胞发育缺陷的irf8突变体。初步研究结果提示irf8突变体的纯合子可以存活，并表现出髓系白细胞发育的缺陷，主要为粒细胞的大量增生。Irf8突变体的表型与临床的髓系白血病表型相似，提示了基于irf8突变体的纯合子建立斑马鱼髓系白血病的可能性。同时，结合已有的基于pu.1G242D突变体构建的M2型髓系白血病模型，和能够活体实时观测白细胞发育状况的Tg(coro1a:eGFP;lyz:Dsred)，高通量的小分子化合物筛选势在必行。本研究将为进一步解析白细胞发育和白血病发生发展的机理并寻找治疗白血病的有效小分子药物提供富有价值信息和参考。

白血病是极为严重的一类血液系统的恶性肿瘤，危害极大。调控白细胞前体增殖、分化和成熟的众多因子的突变是导致白血病发生的重要因素。IRF8是较为常见在白血病进程中扮演重要角色的调控因子。临床研究表明约79%的慢性髓系白血病(CML)和66%的急性髓系白血病(AML)病人表现为IRF8水平的降低或缺失。IRF8功能缺失的小鼠会患慢性和急性髓系白血病。但对于IRF8调控髓系白血病的分子机制仍知之甚少。.通过对稳定遗传的irf8Δ57/Δ57和irf8Δ11/Δ11突变体的表型分析，结果表明irf8突变体中肾脏血的前体细胞群体大量增生，这些前体细胞中大部分都表现为未分化的blast cell状态，irf8突变体的成鱼会表现出髓系白血病的表型。因此，基于irf8突变体的髓系白血病模型成功创建。进一步研究表明，Mertk-ERK信号通路在irf8突变体中过度活化。在髓系细胞中过表达Mertk也会使斑马鱼出现髓系白血病的表型。且当Mertk及ERK功能受到抑制时，irf8突变体的白血病症状会出现明显缓解。因此，Irf8-Mertk-ERK信号通路在髓系白血病发生中作用重大。同时，对于irf8Δ57/Δ57/pu.1G242D/G242D斑马鱼进行细致分析发现irf8和pu.1功能同时缺失时白血病的发生进程加快且表型更加严重。因此，IRF8和PU.1协同调控着髓系细胞的发育并阻止白血病的发生。整个研究进一步扩宽了学界对于IRF8调控髓系白血病发生机制的认识，为治疗白血病提供了新视角。.在课题执行中，建立了“阿片类药物引起的肠功能紊乱疾病（Opioid-Induced Bowel Dysfunction (OIBD)”斑马鱼模型，并发现这种症状的产生源于乙酰胆碱信号通路活性的抑制。此项研究成果发表在《Scientific Reports》。同时，本研究还成功建立了斑马鱼幼鱼的严重细菌感染模型，解析了严重细菌感染导致的髓系应急造血过程。进一步揭示了Pu.1在正常和应急状态下髓系细胞发育中的作用。整个研究创建了严重细菌感染模型并初步揭示了分子机制。研究成果发表在《Scientific Reports》。