RHEB对脐血造血干细胞的影响及调控机制

Umbilical cord blood (UCB) is gradually becoming a valuable source of hematopoietic stem cell (HSC) for transplantation. Compared with bone marrow and peripheral blood, HSC in UCB is more primitive and the proliferative potential is much higher. HSC expansion and engraftment efficiency are attracting more and more attentions because of the limited HSC absolute number of individual UCB. HSC self-renewal and differentiation are regulated by multiple internal and external cues. However, the regulatory mechanism is still unclear. Our previous study showed Rheb deletion impairs self-renewal and differentiation of HSCs both in murine fetal liver and adult bone marrow. However, the role of RHEB in human HSC and its regulatory mechanism is still unclear. Here we propose to manipulate RHEB expression in UCB HSCs, and use 1) in vitro differentiation model to explore the effect of RHEB on HSC proliferation, apoptosis, differentiation and migration; 2) in vivo transplantation assay to study the effect of RHEB on HSC self-renewal, differentiation, migration and homing capacity; 3) RNA-seq and bioinformatic analysis, and molecular biochemistry assays to delineate the regulatory mechanism of RHEB in HSC. This could provide a theoretical basis to some extent for clinical applications of UCB HSC for HSCT.

脐血是造血干细胞移植（HSC transplant, HSCT）供体的重要来源，与骨髓、外周血相比，脐血HSC更原始且增殖能力更强，但是单份脐血HSC数量少，因此扩增HSC数量及提高其植入率广受关注。HSC自我更新和分化功能的维持受细胞及其微环境调控，但具体机制仍不清楚。已知mTOR信号通路在维持HSC功能中至关重要，Rheb可正向调控mTOR，也可独立于mTOR调控细胞生命活动。我们前期研究发现Rheb缺失导致小鼠胎肝和成年鼠HSC自我更新和分化功能受损，但RHEB在人HSC中的作用及机制仍不清楚。本课题以脐血HSC为研究对象，改变RHEB表达后，利用1）体外诱导分化模型研究RHEB对HSC增殖、凋亡、分化和迁移的影响；2）体内移植来研究RHEB对HSC自我更新、分化、迁移和归巢的影响；3）基因芯片和分子生物学方法明确RHEB调控HSC的机制，为临床脐血HSCT的应用提供一定的理论基础。

脐血是造血干细胞移植（HSC transplant, HSCT）供体的重要来源，与骨髓、外周血相比，脐血HSC更原始且增殖能力更强，但是单份脐血HSC数量少，因此扩增HSC数量及提高其植入率广受关注。HSC自我更新和分化功能的维持受细胞及其微环境调控，但具体机制仍不清楚。已知mTOR信号通路在维持HSC功能中至关重要，Rheb可正向调控mTOR，也可独立于mTOR调控细胞生命活动。我们前期研究发现Rheb缺失导致小鼠胎肝和成年鼠HSC自我更新和分化功能受损，但RHEB在人HSC中的作用及机制仍不清楚。我们发现RHEB在人造血干祖细胞的表达明显高于其他造血各系细胞，在脐血CD34+细胞表达明显高于CD34-细胞。本课题以脐血HSC为研究对象，敲降RHEB表达后，1）CD34+细胞的细胞周期与调亡没有明显变化。2）HSC的归巢能力没有明显变化。3）体外克隆实验显示敲除RHEB，CD34+细胞克隆形成的总数是降低的，CFU-GM和CFU-E的数目增多。这些为临床脐血HSCT的应用提供了一定的理论基础。