用于高通量药物筛选的斑马鱼髓系白血病模型的建立

The classical drug discovery approach, is a screen for small molecules that bind in vitro in cell lines. This approach involves screening a large library of compounds to find small molecules that disrupt a phenotype in a biological assay. Whole organisms offer several advantages over cell lines for drug screens, providing information on tissue specificity, toxicity, and accounting for bioavailability. Furthermore, cells are not transformed and are in their normal physiological enviroment of cell-cell and cell-extracellular matrix interactions. The fact remains that mouse embryos require the uterine environment and are more expensive to produce in large numbers for whole mount large-scale drug screens, while zebrafish-based drug screening combine the advantage of high throughput, simple drug administration, and durg targeting in vivo which now has been used extensively for primary drug screening. Myelopoiesis is tightly controlled by interaction of transcription factors such as PU.1, CEBPα and RUNX1, one of whose impaired function can contribute directly to Chronic Myeloid Leukemia (CML). However,none ideal zebrafish model was provided to have a deep insight of CML..Through reverse genetic screening, we obtained a partial loss-of-function pu.1 zebrafish mutant with a point mutation in the DNA binding domain of pu.1 gene. The mutant adult fish had distended head and splayed eyes due to retro-orbital infiltration by malignant cells. The mutation caused abnormal differentiation of myeloid progenitor and significant increase of granulocyte in the early stage. We found the percentage of granulocyte in the kidney marrow and peripheral blood is significant higher in the mutant than in wild-types. We confirmed that pu.1 mutation can leads to an accumulation of the immature myeloid blast cells in zebrafish, which similar as human CML phenotype..In this proposed research, we aim (1)to identify phenotype of the pu.1 mutant; (2) to establish an ideal zebrafish luekemia model with pu.1 mutant and investigate its pathogenesis, typology, clinic data comparison; and (3) to verify the model using some known clinic drugs. These study will further deepen our mechanistic understanding of myeloid lineage development in general and the CML animal models will provide new melecular markers for early diagnosis, prognosis, treatment effect evaluation and new strategies for drug discovery and therapeutic treatments targeting CML.

与细胞和小鼠模型相比，斑马鱼整体高通量药物筛选具有高通量、给药、生物和靶点等优势，已成为发现候选药物的核心技术并被广泛应用。髓系造血是通过各种转录因子如PU.1、CEBPα和Runx1等相互作用实现的，其调控缺陷可导致髓系白血病的发生。但目前还缺少一个理想的斑马鱼模型。.我们前期采用化学诱变和反向遗传学筛选技术获得一个pu.1基因点突变的斑马鱼突变体，具有以下初步表型：①早期随系祖细胞分化异常，粒细胞明显增生；②成鱼眼睛突出、头顶部膨起似组织浸润；③成鱼外周血和肾脏中粒细胞比例明显增多，髓系发育停留在幼稚期等。我们假设斑马鱼pu.1突变体有可能是一种慢性粒细胞白血病的动物模型。.本项目拟进行：①突变体表型鉴定；②斑马鱼白血病模型的建立及其发病机制、分型、临床比对；③已知药物对斑马鱼模型的药理学验证，以期获得新的白血病发病机制、临床诊断预后和治疗评估的分子标记物和药物筛选平台。

本项目我们发现抑制斑马鱼Pu.1的功能会导致斑马鱼髓系细胞的扩增。Pu.1是Ets转录因子家族中的一员，在早期髓系祖细胞产生过程中必不可少，是决定髓系向单核细胞还是向粒细胞分化的重要因子。据报道Pu.1还参与人与小鼠的白血病生成过程，是髓细胞白血病的抑癌基因。通过TILLING的方法，我们获得了一个pu.1表达减少的突变体pu.1G242D。运用遗传学和细胞学的方法研究了突变体斑马鱼pu.1G242D定向造血早期以及成鱼中中性粒细胞的分化。结果表明在pu.1G242D突变体中不成熟的中性粒细胞在早期尾部造血组织CHT区（相当于哺乳动物胎肝的造血器官）以及成鱼的肾脏血（相当于哺乳动物的骨髓）中增多。在18个月突变体的外周血以及肾脏血中出现原始髓系细胞的累积。细胞化学实验进一步证明了这些增多的中性粒细胞是由于过度增殖而不是凋亡减少产生的。pu.1G242D突变体的血液表型和人类的骨髓增生异常综合症（MDS）和急性髓系白血病（AML）类似，并且对抗肿瘤药物阿糖胞苷敏感。因此，我们的研究证实了抑制Pu.1的功能会导致斑马鱼髓系细胞发育失调，并且提供了pu.1G242D突变体作为一种有价值的药物筛选和评估模型。[Leukemia.2013.27(9):1913-7]、[Sci China Life Sci. 2015 Dec;58(12):1202-8. doi: 10.1007/s11427-015-4965-6. Review]、[授权公告号：CN103977424B, 2016]

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