小鼠精母细胞I-SceI报告系统的建立及在DNA双链断裂同源重组修复研究的应用

Infertility is a major health problem that affects approximately 15% of couples. Although many advances have been made in the treatment of male infertility, the molecular and genetic causes of male infertility remain largely elusive. The process of meiotic recombination is essential for fertility and integrity of the genome. Altered recombination may result in various sperm abnormalities, such as arrest in spermatogenesis or non-disjunction, which in turn contributes to infertility or aneuploidy. Chromosomal crossover often begins when a protein called SPO11 makes a targeted double-strand break in DNA. Meiotic recombination events are distributed unevenly throughout eukaryotic genomes including hotspots and coldspots, which brings great challenge to molecular mechanism research of homologous recombination-mediated repair of DNA double-strand breaks. In this study, we construct an I-SceI reporter system in mouse spermatocytes to induce a site-specific DNA double-strand breaks, and apply the cell model and animal model to signal transduction involved in homologous recombination-mediated repair of DNA double-strand breaks. The I-SceI reporter system in mouse spermatocytes could provide us with a new and practical tool for elucidating molecular mechanism of non-obstructive azoospermia.

不育已经成为一个严重的健康问题，育龄夫妇中发病率高达15%。近年来，男性不育的治疗虽然取得了较大进展，但是其分子遗传学机制仍未阐明。减数分裂同源重组过程与男性生育、基因组的完整性有着密切关系，其异常会导致不同程度的精子异常，如精子发生停滞而出现不育，或精子染色体异常导致非整倍性变异。染色体的交换起始于蛋白SPO11形成DNA双链断裂。减数分裂同源重组具有位点多，而且存在热点和冷点的不平衡，这给DNA双链断裂同源重组修复的分子机制研究带来了巨大挑战。本研究拟通过在细胞和动物中构建精母细胞I-SceI报告系统，产生位点特异的DNA双链断裂，并将其应用到DNA双链断裂同源重组修复信号通路研究。I-SceI报告系统在小鼠精母细胞中的建立有望为特发性男性不育分子机制阐明提供新的有效的研究工具。

不育已经成为一个严重的健康问题，育龄夫妇中发病率高达15%。近年来，男性不育的治疗虽然取得了较大进展，但是其分子遗传学机制仍未阐明。DNA双链断裂及修复途径与染色体异常和男性不育密切相关。本研究通过构建精母细胞I-SceI报告系统，产生位点特异的DNA双链断裂，并将其应用到DNA双链断裂同源重组修复信号通路及应用研究。.本研究成功构建精母细胞I-SceI报告系统，产生位点特异的DNA双链断裂，并已经将其应用到DNA双链断裂同源重组修复信号通路研究。我们研究发现了新的相关通道蛋白MRE11和MSH2。.我们构建了一个细胞内NHEJ活性分析系统。它通过I-SceI形成DNA双链断裂，监测细胞 NHEJ修复活性。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。.我们建立了一个定量检测基因组中DNA双链断裂数目的方法，并将其用于男性不育患者的SDF分析。.我们研究了染色体易位相关信号通道蛋白，发现了染色体易位相关的分子标志物Ku70、p300和MSH2，为染色体易位的分子机制的阐明提供了新的途径。.我们建立了一系列反映DNA双链断裂标志物的方法，如单细胞凝胶电泳、微核试验、γH2AX、53BP1、MLH1、8-OHdG等，并将其应用于男性不育、环境监测中。