MSH5 C85T多态性与电离辐射所致生精异常个体差异的分子机制研究

Individual variability in radiation-sensitivity is large in spermatogenic impairment. Single nucleotide polymorphisms (SNPs) in genes involved in DNA repair could be responsible for such cases of radiosensitivity. However, the underlying mechanism in this respect is still largely unknown. Studies have shown that DNA repair protein MSH5 is involved in the process of meiosis, and may be also involved in other aspects of DNA damage response. Gene knockout of MSH5 results in sterility in mice. In particular, a recent study suggested that the effect of MSH5 P29S on promoting mitotic apoptosis can be expected to have a mirrored impact on germ cell apoptosis. Our findings indicated that there is an association of polymorphism C85T in MSH5 with male infertility, specifically azoospermia or severe oligozoospermia. Also, MSH C85T polymorphism is associated with sperm integrity and apoptotic ratio before and after radiotherapy in men with cancers. Therefore, we hypothesize that MSH5 C85T(P29S) is involved with spermatogenic impairment in response to ion radiation. In this study, we examine the effects of spermatogenic cells transfected with MSH5 C85T vector established with PCR site-directed mutagenesis on cell survival, apoptosis and DNA damage. In addition, the function of MSH5 in response to ion radiation will be studied in spermatogenic cells. In summary, this study could offer some direct evidences that MSH5 C85T(P29S) has an important role in the risk of spermatogenic impairment in response to ion radiation.

单核苷酸多态性与电离辐射所致机体生精异常的个体差异有关，但其分子机制一直不明。MSH5参与减数分裂及DNA损伤的应答，其基因敲除可导致小鼠不育。对体细胞研究表明MSH5 C85T(P29S)多态性导致电离辐射所引起的细胞凋亡增加。我们的研究也显示MSH5 C85T与少精/无精男性不育有密切关系，肿瘤患者在放疗前后精子数量和凋亡率也与MSH5 C85T多态性相关。因此，我们提出电离辐射所致生精异常的个体差异与MSH5 C85T(P29S)多态性有关。本研究拟用PCR-定点诱变技术构建MSH5 C85T变异体，转染生精细胞，探讨MSH5 C85T(P29S)的改变对辐射诱导的生精细胞存活、凋亡及DNA损伤的影响，并研究其信号通路，揭示MSH5在辐射导致的生精细胞异常的分子机制。本项目的实施将为放射医学的组织细胞差异性应答的研究提供新的途径和方法。

单核苷酸多态性与电离辐射所致机体生精异常的个体差异有关，但其分子机制一直不明。MSH5参与减数分裂及DNA损伤的应答。对体细胞研究表明MSH5 C85T(P29S)多态性导致电离辐射所引起的细胞凋亡增加。我们的研究也显示它与少精/无精男性不育有密切关系。因此，我们提出假说认为电离辐射所致生精异常的个体差异与MSH5 C85T多态性有关。.我们研究了113例睾丸生殖细胞肿瘤患者放疗前后生精功能，结果显示，睾丸生殖细胞肿瘤患者与对照组间MSH5 CC、CT+TT分布的差异没有统计学意义(P＞0.05)，精液质量参数中的精子计数、精子浓度和DFI差异具有统计学意义（P＜0.05）。结果表明hMSH5的多态性与患者放疗后生精功能的恢复密切相关。.基于PCR-定点诱导技术构建MSH5 C85T载体，并将其转染生精细胞GC-1 spg、GC-2 spd、TM3和TM4，观察不同剂量的辐射对细胞存活、凋亡和DNA损伤程度的影响。结果显示，仅有GC-2 spd细胞对辐射敏感性改变具有统计处意义(P ＜ 0.05)。在GC-2细胞中构建的I-SceI诱导的DNA双链断裂荧光报告系统显示MSH5、MLH1、MSH2和MRE11等蛋白在DSBs修复中起重要作用。.构建DNA双链断裂非同源末端连接（NHEJ）分析载体，通过I-SceI形成DNA双链断裂，监测细胞 NHEJ修复活性，结果显示，转染MSH5变异型的载体的细胞比转染MSH5野生型载体的细胞%NHEJ差异具有统计学意义（P＜0.05）。将转染不同MSH5 C85T载体的细胞进行WB分析，MSH5的表达也存在差异。MSH5 C85T多态性可能通过参与DNA双链断裂的NHEJ修复途径影响其辐射的敏感性。.总之，我们的研究首次表明了MSH5 C85T多态性与电离辐射所致生精异常的关系。MSH5 C85T有望成为精准医疗的一个重要靶点。