PUMA在人造血干祖细胞抗放射作用中的功能及分子机制研究

Bone marrow injury is a major adverse side effect of whole-body radiation or chemotherapy, which directly influences the treatment outcome and life quality of patients. How to minimize the damage of hematopoietic system during radiotherapy and chemotherapy, and restore hematopoietic reconstruction are the most challenging tasks to be solved in transplantation medicine. Puma is a key regulator downstream of P53 signaling pathway. Our perviousstudies show thatdeletion of Puma in mouse confers long-term survival in response to high-dose γ-irradiation and there is no increase of malignancy in the exposed mice. Thus, PUMA may be a potential target in radioprotection. However, how PUMA exerts its function in human hematopoietic stem and progenitor cells are poorly understood. In this project, we will firstly delete PUMA gene in human cord blood CD34+ cells using CRISPR-Cas9 technology to investigate the effect of PUMA gene deletion on the biological function and radioprotective effect of human cord blood CD34+ cells both in vitro and in vivo. To further investigate the molecular mechanism of radioprotection effect of PUMA, we will then determine the function of PUMA in DNA repair pathway. Finally we try to identify the ideal target for tumor radiation therapy and provide theoretical and experimental basis for the application of PUMA in radioprotection.

骨髓损伤是肿瘤放射治疗和化学治疗最主要的副作用，直接影响治疗效果及患者的生存质量。如何减少放疗对造血系统的损伤并加速造血重建恢复是目前肿瘤治疗中亟待解决的问题。我们的前期研究表明，作为P53信号通路下游关键凋亡调控因子，Puma基因敲除显著提高致死剂量γ放射处理后小鼠的长期存活率，并且不增加肿瘤形成的风险，因此Puma可能是潜在的抗辐射靶点，但PUMA缺失对人造血干祖细胞的抗辐射保护作用还不清楚。本课题中，我们将利用CRISPR-Cas9技术在人脐血CD34+细胞中敲除PUMA基因，然后在体外和NOD/SCID免疫缺陷小鼠体内研究PUMA基因敲除后是否对人的造血干祖细胞产生抗放射保护作用，同时探索PUMA敲除抗放作用的分子机制。该研究将为临床抗放射治疗找到新的靶点，并为进一步开发靶向PUMA的抗辐射药物提供理论与实验依据。

该项目按照研究计划执行，建立并优化利用CRISPR-Cas9在多种细胞系中的基因敲除和敲入系统。利用优化的方法，在人原代T细胞和造血干祖细胞中的基因敲除效率达到80%以上，精确基因插入效率可达20-50%，这为进一步开展Puma基因功能的研究工作打下基础。利用对CRISPR-Cas9基因编辑效率的优化，我们构建了PUMA敲除的细胞系：主要构建了PUMA敲除的293T细胞、PUMA敲除的iPS细胞。结果表明，PUMA敲降后，可以显著提高CRISPR-Cas9系统下293T和iPS的编辑效率，即可以提高CRISPR-Cas9系统中同源重组修复的比例。在Puma敲除的小鼠造血干祖细胞中我们也同样观察到了这一现象，说明Puma的抗放射作用是由于影响同源重组修复过程产生的，初步阐明了Puma的抗放射作用的主要机制。