ND融合基因促进造血干细胞扩增的机制研究

Losing stemness and differentiation are common phenomena during ex vivo culture of hematopoietic stem cells (HSC),which limits the translational application of HSC from cord blood and other resources.Enforced expression of artificial Nup98-Hoxa10HD (ND) fusion gene in HSC can expand HSC over 1000 folds within 10 days, under defined culture conditions. Of note, HSC expanded by ND produce normal hematopoiesis in vivo. We hypothesize that the targeted genes and pathways of ND form an intrinsic module, which is essential to HSC expansion in vitro. Manipulating the ND effector module might help to expand HSC in large scale through long-term culture in vitro. Based on our preliminary work, we plan to dissect the intrinsic regulating mechanism of HSC expansion in vitro, through building an expanding-HSC model. Then, with the advantage of the expanding-HSC model, RNA-seq and CHIP-seq approaches will be applied to investigate the underling mechanism. Key candidate effectors correlated to the expanding consequence by ND will be functionally evaluated in terms of HSC expansion. Our research might provide molecular and signaling targets for manipulating and expanding HSC ex vivo, which is important for translational application of HSC.

体外无法有效扩增造血干细胞(HSC)是包括脐血干细胞移植在内HSC转化医学的关键问题。仅靠细胞外因素扩增HSC会出现HSC干性下降及增殖分化的现象，无法达到扩增预期。 研究发现，Nup98-HoxA10HD (ND) 人工融合基因在HSC中异位表达，可在明确的体外培养条件下扩增HSC达1000倍以上，扩增后HSC具有正常生理造血功能。ND扩增HSC的机制尚不明确。本研究拟构建ND条件性基因敲进小鼠模型，从骨髓分离原代HSC，体外扩增建立HSC扩增期细胞模型。基于此独特模型，利用RNA-seq、Chip-seq等高通量组学分析技术，结合一系列基因表达调控技术，骨髓移植等HSC功能评估方法，在分子、细胞和动物模型水平上深入研究ND促进HSC扩增的内在信号调控机制。本研究将加深对HSC体外自我更新调控的理论认识，为开发体外操控、扩增HSC新技术提供靶分子，对HSC转化医学研究具有重要意义。

造血干细胞(HSC)体外培养容易产生自我更新能力下降、丢失以及增殖性分化等现象。Nup98-Hoxa10HD人工融合基因(原简称ND基因修改为NA基因)通过病毒感染实现在HSC中异位表达可以促进HSC体外扩增达1000 倍以上，扩增后的HSC依然维持正常生理造血功能。本研究构建了NA条件性基因敲进小鼠模型，并进行了一系列骨髓移植实验、体外维持实验、体外扩增实验表明，遗传途径NA修饰的HSC具备正常造血生成能力、移植竞争力增强、可以体外长期维持、实现体外扩增培养。本项目还比较了NA、NrasG12D、NAV（NA与NrasG12D复合）三种基因型HSC的移植竞争力发现NAV HSC具有最强的竞争优势，且NAV HSC单个细胞能够在无滋养层的体外培养条件下存活、扩增，培养后依然能够支持造血重建。结合RNA-seq和基因表达调控技术，本研究还进一步发现了NA调控HSC的差异分子特征。本研究将加深对HSC体外自我更新调控的理论认识，为开发体外操控、扩繁HSC新技术提供了潜在靶分子，对HSC转化医学研究具有重要意义。