UTF1基因在少弱精症及非梗阻性无精子症中的作用及分子机制

Although a great progress has been made in the treatment of infertility, but now there still lacks of effective treatments on oligospermia, asthenozoospermia and non-obstructive azoospermia. Spermatogonial stem cells (SSCs) are seed cells that can proliferate and differentiate to sperm, which have potential clinical value in infertility treatment. The process of proliferation and differentiation to sperms is regulated by network of multiple genes. To find out the intersection gene and common gene is a feasible way for studying on oligospermia, asthenozoospermia and non-obstructive azoospermia. UTF1 gene plays an important role in maintaining proliferation of SSCs, so it is speculated that changes of UTF1 in expression, epigenetics and SNP are one of the causes for oligospermia, asthenozoospermia and non-obstructive azoospermia. Based on our previous studies on surface marker, isolation, purification, in vitro culture, cryopreservation and transplantation of SSCs, SSCs are intended to be the starting point. In addition, a lot of technologies and methods (gene chip, epigenetics, gene sequencing, SSCs transplantation into seminiferous tubules by microinjection, fluorescence tracking analysis) are used to study the correlation of UTF1 gene with oligospermia, asthenozoospermia and non-obstructive azoospermia and its regulatory mechanism, and elucidate the role of UTF1 gene in the process of SSCs proliferation, differentiation and development of sperms. It provides a new thought and strategy for the causes of oligospermia, asthenozoospermia and non-obstructive azoospermia as well as gene diagnosis and gene targeting therapy.

尽管在不育治疗方面取得较大进步，对一些少弱精症或非梗阻性无精子症（NOA）目前仍缺乏有效治疗方法。精原干细胞（SSCs）作为增殖分化形成精子的种子细胞，在不育治疗方面有潜在的临床应用价值，其增殖分化形成精子的过程受多基因参与的网络调控，寻找主要的交点基因和共同作用基因是研究少弱精症、NOA发生的可行思路。UTF1基因在维持SSCs增殖方面具有重要作用，推测UTF1基因在表达、表观遗传、基因SNP方面的变化是引起少弱精症、NOA的原因之一。我们在前期对SSCs表面标志物、分离纯化、体外培养、冷冻保存及移植研究的基础上，拟以SSCs作为切入点，通过基因芯片、表观遗传、基因测序、曲细精管微注射移植、荧光追踪分析等技术，研究UTF1基因与少弱精症、NOA发生的相关性及其调控机制，阐释UTF1基因在SSCs增殖分化及精子发生中的作用，为少弱精症、NOA的致病原因、诊断、靶向治疗提供新的思路与策略。

主要研究内容。.项目主要研究内容如下①体外构建小鼠精原干细胞（SSCs），并通过SSCs移植技术进行验证，②构建人急性淋巴细胞白血病小鼠模型，通过不同SSCs纯化技术分选模型鼠SSCs，通过SSCs移植技术验证不同SSCs纯化技术在肿瘤患者SSCs移植应用的安全性③体外构建人SSCs培养体系，通过慢病毒 UTF1 过表达与 shRNA 抑制技术结合转录组测序分析，明确 UTF1 基因在维持SSCs增殖分化方面的作用及其相关信号调控通路。④通过基因表达、表观遗传、基因测序技术与方法，明确 UTF1基因与临床少弱精症、非梗阻性无精子症发生的相关性及其调控机制。.主要结果①小鼠精原干细胞体外培养体系建立及功能鉴定，本项研究中建立的小鼠SSCs体外培养体系不仅能够促进SSCs大量增殖，同时保持了其未分化SSCs的干细胞特性。.②多种精原干细胞纯化方法在精原干细胞移植技术中应用的安全性比较：结果发现接受密度梯度离心分选纯化的精原干细胞移植的小鼠均诱发了急性B淋巴细胞白血病，而移植了经免疫磁珠及流式细胞仪分选纯化的精原干细胞的受体小鼠均未诱发急性B淋巴细胞白血病，本项研究结果为肿瘤患者进行精原干细胞冻存-移植生育力保存应用提供了理论依据。③人精原干细胞体外培养体系的建立：通过本体系构建的人SSCs培养体系能够在体外快速扩增形成高度纯化的SSCs，同时保持较好的生殖干细胞特性，可用于SSCs移植恢复生育力。④UTF1基因调控人精原干细胞增殖中的作用及其机制：UTF1基因通过调控HSA04110通路多种基因表达进而调控人SSCs增殖，是人SSCs增殖重要调控基因。