【作者】 陈陟阳；

【导师】 杨志伟； 鞠振宇；

【作者基本信息】 北京协和医学院 ， 生理学， 2011， 硕士

【摘要】 WIP1(Wild-type p53-induced phosphatase)是一癌基因,在众多癌症细胞中高表达,同时是p53负反馈调节环路的重要组成成分,参与调控细胞的稳态平衡。为了研究WIP1基因与骨髓造血干细胞(Haematopoietic stem cells,HSCs)的维持和再生的关系,我们通过WIP1基因敲除小鼠的体外HSC单克隆培养、体内竞争性移植等实验研究WIP1在HSC自我更新能力和多系分化能力中的作用。实验结果显示WIP1基因缺失小鼠的骨髓B细胞比例下降,胸腺T细胞发育障碍；在干细胞和祖细胞水平,WIP1基因缺失小鼠的长期/短期造血干细胞增多,多潜能祖细胞及淋系共同祖细胞比例下降；体外单克隆培养实验显示,WIP1基因缺失HSC形成克隆能力下降；全骨髓及干细胞竞争性移植实验和连续移植实验显示,WIP1基因缺失小鼠骨髓长期造血干细胞造血重建能力明显低于野生型；5-FU处理实验显示,WIP1基因缺失的HSC对5-FU敏感性增强；在半致死剂量辐射损伤情况下,WIP1基因缺失的小鼠长期造血干细胞的比例进一步增加；8.5Gy放射线照射损伤实验显示,WIP1基因缺失的HSC对放射线敏感性增强；移植实验显示WIP1基因缺失小鼠HSC数目增多及功能下降的表型不依赖于HSC微环境及整体环境；WIP1缺失小鼠HSC功能下降不依赖于p38及p38介导的氧化应激信号通路。这样研究结果显示,无论在正常情况下还是应激反应中,WIP1基因缺失均导致HSC数目增加但是再生能力下降,说明WIP1基因在维持HSC稳态方面起重要作用。更多还原

【Abstract】 WIP1 (or PPM ID), Wild-type p53-induced phosphatase 1, is recognized as an oncogene, displaying high expression level in cells of multiple cancer types. Meanwhile, it is also known as a very important component in p53 negative feedback pathway, hence plays a vital role in regulating homeostasis of cells. In this study, we aimed to examine the effects of WIP1 gene on self-renewal and differentiation of hematopoietic stem cells (HSCs) in both normal and stress situations. To this end, HSCs of WIP1 gene knockout mouse were evaluated by in vitro clonal assay and in vivo tranplantation expriment..Our data showed that the total number of B cell in bone marrow was decreased although the proportion of different subsets in B cell development was normal; in thymus development, the proportion of CD8/CD4 double negative cells was increased while the CD8/CD4 double positive cells was decreased. We also found an increased cell number in the stem cell compartment but decreased cell numbers at the multipotent progenitors and common lymphoid progenitor level in Wipl gene knockout mice bone marrow,. The in vitro single-cell clonal assay and the in vivo bone marrow transplantation experiment showed that Wipl gene knockout murine HSC has defect in forming big size colony and reconstituting the recipient hematopoietic system. Under replicative stress and the irradiation stress conditions, Wipl(?) murine HSC frequencies were further increased compared to the littermate controls. However, the Wipl(?) HSCs showed increased sensitivity to myelotoxic stress and high dose irradiation. Further expriment showed that these phenotype was due to HSC cell intrinsic defect but not dependent on the defect of HSC microenvironment. Moreover, the defect of WIPl(?) HSCs cannot be rescued by blocking p38 signal pathway or antioxidative treatment. Thus, our results demonstrate that Wipl plays a pivotal role in regulating the HSC maintenance and function.更多还原