利用斑马鱼模型研究kri1蛋白在造血干祖细胞发育中的作用

Hematopoietic stem cells (HSCs) give rise to all the lineages of blood cells in human body. The development, self-renewal and differentiation of HSCs are strictly and accurately controlled in embryonic development and also in adulthood. Disordered regulatory mechanisms results in types of blood disease, like anemia or leukemia. To elucidate such important regulatory network, we performed a large-scale forward genetic screen with HSC markers on zebrafish model organism. We found ldd499 mutants zebrafish were overall morphological normal, but carrying HSCs increasing phenotype as homozygotes. Through genetic mapping and subsequent validation, we confirmed ldd499 phenotype is due to the loss of kri1 protein, caused by nonsense mutation in its genomic coding region. This project aims to understand Kri1-mediated regulation on HSCs development. With the combination of genetic and developmental biology approaches, we plan to study on the role kri1 in HSCs determination. Furthermore, we will perform molecular and biochemical experiments to reveal its mechanism. Through genomic analysis on human patients blood samples, certain type of anemia or leukemia will be linked with kri1-regulatory mechanism and with potential clinical applications.

造血干细胞是机体中所有成熟血细胞的源头。造血干细胞的发育、自我更新以及分化的机制是受到严格精确调控的，如果这种调控机制出现异常会产生严重的血液疾病，如贫血或白血病。为了充分阐述这一调控网络，我们利用斑马鱼模式生物开展了定向造血表型的遗传学筛选。在突变库中我们发现了家系ldd499表现为造血干祖细胞的减少。目前已经完成对突变体表型的分析和定位克隆，并确定是由于kri1基因的失活所造成的造血干祖细胞的减少表型。本课题的目标是揭示kri1蛋白调控造血干细胞发育的机理。我们将一方面利用斑马鱼模型明确kri1失活对造血干细胞发育的细胞学和发育生物学水平的影响，另一方面在分子水平阐述kri1调控造血干祖细胞发育的机制，希望此项研究为诊治人类疾病提供重要理论依据。

造血干细胞作为成体干细胞的一种是体内所有成熟血细胞的来源，对维持机体的正常生理功能有重要作用。深入研究和解析造血干细胞自我更新和分化的机制可为相关血液疾病如贫血或白血病的诊治提供重要理论依据。前期通过在斑马鱼中进行的大规模前向遗传学筛选，我们得到了一个造血干祖细胞减少的突变家系cas002，类似于人类的贫血表型。定位克隆和morpholino 敲低等技术证实 kri1l 的突变引起了该表型。Kri1 的突变引起了18S rRNA的大量减少，核糖体发育异常、总蛋白量减少及造血干祖细胞数量减少。不同于以往核糖体相关的贫血疾病是通过激活p53通路引起凋亡的机制，在突变体中抑制p53通路并不能缓解贫血的症状，而过表达 bcl2 能够很好地恢复造血衰竭的表型。通过生化检测和活体观察，我们发现突变家系的造血干祖细胞中自噬过度增加，而过表达bcl2可以通过抑制自噬，恢复贫血表型。同时自噬的小分子抑制剂（3-MA，BafA1，chloroquine）能够有效恢复造血干祖细胞衰竭和成熟细胞减少的表型。本研究发现了核糖体异常引起的贫血表型和过度自噬的新联系，为核糖体相关疾病的机制研究提供了新的方向，同时为核糖体异常引起的贫血疾病诊治提供了新思路。