目的： Grwd1（Glutamate Rich WD Repeat Containing 1）与多种疾病的不良预后以及生 存期相关，在实体瘤中该基因已经被证实为一种新的癌基因，其作用机制与调控 p53 通路以及参与 DNA 复制装载有着密切联系。目前在血液系统疾病相关模型中也发 现该基因的异常表达，但其发挥的功能以及具体机制尚不十分明确。本研究通过在 小鼠骨髓细胞系 32D 以及 c-Kit 等细胞中构建 Grwd1 表达异常的模型，探究 Grwd1 对造血细胞的生物学功能影响以及其相关机制。

方法： 在小鼠骨髓细胞系 32D、c-Kit 细胞以及伴有髓系肿瘤相关基因异常的 32D，通 过慢病毒转染的方法建立 Grwd1 低表达的细胞模型。检测不同细胞中，Grwd1 降低 后对细胞增殖能力、细胞周期、细胞凋亡以及集落形成能力等细胞功能的影响。进 一步利用蛋白印迹实验以及实时荧光定量 PCR 初步验证 Grwd1 的调控机制，明确 该基因对 DNA 损伤刺激的敏感性变化。

结果： 成功构建了 Grwd1 敲降的 32D 以及 Asxl1-/- 32D 细胞系。细胞功能学实验证实， Grwd1 的敲降导致 32D 细胞出现周期阻滞，G0-G1 期细胞比例增加、集落形成能力 减弱等现象；小鼠原代细胞中敲降 Grwd1 可出现髓系偏向分化的现象； Asxl1-/- 32D 则表现出抑制增殖、恶性造血增加、DNA 损伤敏感性增高的现象。机制探究发现， Grwd1 的敲降导致生存压力刺激下 P53 蛋白的含量增加，p53 通路下游基因表达增 加。

结论： Grwd1 的敲降影响 32D 细胞生物学功能，但不影响细胞的存活。而 Grwd1 的 敲降可加重 Asxl1 缺失的 32D 细胞的核异常现象，显著减慢其增殖速率及集落形成 能力，并导致小鼠原代 c-Kit 细胞出现髓系偏向分化的情况。这些现象主要是因为Grwd1 的低表达可以影响 P53 蛋白的稳定性，从而增加 P53 蛋白的含量并激活 p53 下游信号通路基因的表达。

Objective: Grwd1 (Glutamate Rich WD Repeat Containing 1) is associated with poor prognosis and survival of a variety of diseases. This gene has been confirmed as a new oncogene in solid tumors, and its mechanism of action is closely related to the regulation of p53 pathway and its involvement in DNA replication loading. At present, the abnormal expression of this gene has also been found in blood disease related models, but its functions and underlying mechanisms are not very clear. In this study, the abnormal expression model of Grwd1 was constructed in normal mouse bone marrow cell lines and c-Kit positive cells to investigate the biological function of Grwd1 on normal hematopoietic cells and its related mechanisms.

Methods: A series of cell lines with Grwd1 knockdown in normal bone marrow cell lines, c-Kit cells and MPN-like cell lines cells were established through lentivirus transfection, and relevant experiments were conducted to detect the effects of Grwd1 knockdown on cell proliferation, cell cycle progression, apoptosis, and colony formation ability of the experimental group. Subsequently, western blot and real-time quantitative PCR were used to verify the intrinsic regulatory mechanism of Grwd1, clarify the sensitivity changes of this gene to DNA damage stimulation.

Results: The 32D and Asxl1-/- 32D cell lines with Grwd1 knockdown were successfully constructed. Cell functional experiments confirmed that Grwd1 knockdown resulted in cell cycle arrest, increased proportion of G0-G1 phase cells and decreased colony formation ability at 32D cells. In primary mouse cells, knockdown of Grwd1 can induce biased myeloid differentiation. While Asxl1-/- 32D showed inhibition of proliferation, increased hematopoietic malignity and increased sensitivity to DNA damage. Mechanism exploration revealed that Grwd1 knockdown resulted in increased P53 protein content and increased p53 pathway downstream gene expression under survival stress. 

Conclusion: Grwd1 knockdown affected the biology function of 32D cells, but did not affect cell survival. However, Grwd1 knockdown can aggravate the nuclear abnormality of Asxl1- deficient 32D cells, significantly slow down their proliferation rate and colony formation ability, and lead to myelogenic differentiation of primary mouse c-Kit cells. These phenomena are mainly because low expression of Grwd1 can affect the stability of P53 protein, thereby increasing the content of P53 protein and activating the expression of p53 downstream signaling pathway genes.