Ts65Dn小鼠造血干细胞对辐射诱导DNA损伤修复障碍研究

【作者】 王营营；

【导师】 孟爱民； 周道洪；

【作者基本信息】 北京协和医学院 ， 放射医学， 2014， 博士

【摘要】 唐氏综合症(Down Syndrome,DS)是人类常见的染色体疾病,患者有三条21号染色体,伴有肌肉、骨骼和神经发育异常,幼年患儿往往表现为造血系统异常,罹患急性巨核细胞白血病的几率增高。Ts65Dn小鼠与人类21号染色体有104个同源基因,与人类患者有相似的病理特症,例如学习和行为障碍,心脏病等,是目前常用的唐氏综合症模型。研究表明非整倍体可能降低DNA损伤修复功能,引起基因组的不稳定性。唐氏综合症相关的造血系统异常和恶性肿瘤可能与造血干细胞(HSCs)自我更新能力受损,造血系统基因不稳定有关,为此我们利用唐氏综合症模型小鼠对Ts65Dn小鼠造血干细胞数量、功能及辐射诱导DNA损伤修复机制进行研究。研究发现与野生型对照小鼠(Wild type,WT)相比,Ts65Dn小鼠的骨髓造血干细胞HSCs数量明显减少,造血祖细胞(HPCs)水平没有明显变化；体外单细胞克隆实验结果显示Ts65Dn小鼠HSCs单细胞克隆形成率明显低于对照小鼠,表明Ts65Dn小鼠骨髓HSC数量减少、功能下降。利用γH2AX表达检测Ts65Dn小鼠HSC/HPC损伤DNA双链断裂(DSB),发现Ts65Dn小鼠γH2AX表达水平高于对照小鼠；体外2Gyγ射线照射后1小时,两种小鼠的HSCs的DSB水平没有差异,但是随着时间延长,照射后3小时和6小时后Ts65Dn小鼠的HSCs的未修复的DSB水平显著高于对照组小鼠。且照射后Ts65Dn小鼠HSCs的克隆形成能力显著降低。与对照小鼠相比,Ts65Dn小鼠造血祖细胞体外克隆能力和DSB水平以及照射后DSB修复能力没有显著差异。结果提示Ts65Dn小鼠在HSC水平出现了DNA损伤修复障碍及功能损伤。USP16基因编码一种去泛素化酶,Ts65Dn小鼠因多余的基因拷贝表达过高的,USP16基因,可能是造血干细胞DSB修复缺陷的重要分子机制。应用RT-PCR两个不同探针检测发现,与WT相比,Ts65Dn小鼠HPC内USP16表达分别升高1.45倍和1.53倍。而HSC内USP16表达分别升高2.21倍和2.04倍。由于Ts65Dn小鼠HSC中USP16过高表达会引起H2A和H2AX的过度去泛素化,干扰双链断裂位置修复蛋白乳腺癌1号基因(BRCA1)复合体,p53结合蛋白(53BP1)等分子的招募,这可能与Ts65Dn小鼠HSC出现DSB修复障碍有关。综上,我们应用Ts65Dn小鼠作为唐氏综合症动物研究模型,发现Ts65Dn小鼠的造血干细胞出现数量和功能的异常,辐射敏感性增加；Ts65Dn小鼠HSC出现DSB修复障碍,可能与USP16在Ts65Dn小鼠HSC中高表达有关。DSB的正常修复是维持基因稳定性的重要因素。本项研究结果表明Ts65Dn小鼠造血干细胞DSB修复障碍可能是唐氏综合症病人早年造血系统异常以及白血病高发的原因之一。USP16有可能成为唐氏综合症治疗中的潜在靶点。更多还原

【Abstract】 Down syndrome (DS) is the most common chromosomal abnormality in humans caused by the presence of all or part of a third copy of chromosome21. In addition to musculoskeletal and neurodevelopmental abnormalities, pediatric patients with DS also display various hematologic disorders and are at increased risk of acute lymphoblastic leukemia and acute megakaryocytic leukemia. Ts65Dn mice are trisomic for104orthologs of the genes on human chromosome21and are one of the most widely used mouse models for DS research, because they exhibit various deficiencies similar to DS patients. It has been shown that aneuploidy can impair DNA damage repair and induce genomic instability. Therefore, we investigated whether hematopoietic stem cells (HSCs) from Ts65Dn mice are deficient in the repair of DNA double-strand breaks (DSBs), because the deficiency in the repair of DSBs in HSCs can potentially contribute to DS-associated hematological abnormalities and malignancies by impairing HSC self-renewal and inducing hematopoietic genetic instability. Our results showed that Ts65Dn mice had significantly less bone marrow HSCs than wild-type (WT) littermate controls, whereas the levels of bone marrow hematopoietic progenitor cells (HPCs) were similar. The lower level of HSCs in Ts65Dn mice was associated with a significantly higher level of DSBs as determined by γH2AX staining and lower level of clonogenic activity in single cell cultures with SCF, TPO and FL3ligand when compared to the cells from WT controls. Although levels of DSBs in HSCs were similar at1hr after exposure to2Gy y-irradiation in vitro, HSCs from Ts65Dn mice had significantly higher levels of unrepaired DSBs than the cells from WT mice at3and6hr after irradiation. Moreover, after exposure to2Gy y-irradiation in vitro, the reduction in clonogenic function was significantly greater in Ts65Dn HSCs than WT HSCs. In contrast, no significant differences in these parameters were observed in HPCs from Ts65Dn and WT mice with or without irradiation.The molecular mechanism by which extra copy of chromosome21causes HSC defect in repair of DSBs remains to be elucidated. It may be attributable to an increased expression of USP16in Ts65Dn HSCs, because USP16is a deubiquitinase that can regulate DSB repair via the Ring finger protein8-Ring finger protein168(RNF8-RNF168) pathway. These findings suggest that an additional copy of genes on human chromosome21can selectively impair the ability of HSCs to repair DSBs, which may contribute to DS-associated hematological abnormalities and malignancies.更多还原