人精原干细胞自我更新与分化为精子细胞的分子机理研究

Spermatogenesis is a process by which spermatogonial stem cells self-renew and differentiate into mature sperm. Much progress has been made by the peers and us on uncovering molecular mechanisms regulating spermatogonial stem cells in animal. However, it remains unknown about molecular mechanisms mediating the self-renewal and differentiation of human spermatogonial stem cells. Notably, the mechanisms controlling fate determinations of spermatogonial stem cells may differ between human and animals, due to the fact that cell types and phenotypes of human and animal spermatogonial stem cells are different. Therefore, it is crucial and necessary to conduct this important project with the following aims: 1) to establish human spermatogonial stem cell line and long term culture of human spermatogonial stem cells, which would provide abundant cells for mechanistic research and clinic applications of spermatogonial stem cells in both reproductive and regenerativie medicine; 2) to explore the regulation of microRNAs (miRNAs) Let-7G and Let-7I as well as PI3K/Akt, Ras/ERK1/2 and Smad2/3 signaling pathways on the self-renewal of human spermatogonial stem cells, which could offer novel molecular mechanisms of human spermatogenesis; 3) to induce human spermatogonial stem cells to differentiate into functional and mature sperm, which could provide male gametes for patients with azoospermia; and 4) to unveil the regulation of miRNA-93 as well as Smad1/5/8 and STAT3 signaling pathways on the differentiation of human spermatogonial stem cells into male germ cells and eventually male gametes, which would provide new insights into molecular mechanisms of human spermatogenesis. Imporantly, this project could provide novel molecular mechanisms regulating the generation of human male germ cells. This project is of particular significance because it could offer functional and mature sperm for treating male infertility and it provides novel targets for developing new contraceptive approaches.

精子发生是精原干细胞自我更新与分化为精子的过程。我们和同行对动物精原干细胞调控的分子机制进行了较多研究。然而，人精原干细胞自我更新与分化的分子机理尚未报道。人与动物精原干细胞的调控机制可能不同。因此，本项目开展将如下研究：1）建立人精原干细胞系和长期培养扩增体系，为人精原干细胞的机理研究和临床应用提供大量细胞来源；2）探讨人精原干细胞自我更新的小RNA（miRNA ）Let-7G和Let-7I调控与PI3K/Akt、Ras/ERK1/2、Smad2/3信号转导，为阐明人精子发生机制提供新的分子机理；3）诱导人精原干细胞分化为有功能的精子，为无精症病人提供种子细胞；4）揭示人精原干细胞分化为精子的miRNA-93调控与Smad1/5/8和STAT3信号转导，为阐释人精子生成机制提供新的机理。本项目为揭示人男性生殖细胞生成机制提供新的分子机理、对治疗男性不育、研发新的男性避孕方法都有重要意义。

由于人类精原干细胞的数量较少，体外难于长期培养和扩增由于人类精原干细胞的数量较少，体外难于长期培养和扩增，人类精原干细胞自我更新与分化的分子调控尚不清楚。我们在本项目完成了如下研究内容：1）建立了第一株人精原干细胞系和人原代精原干细胞体外长期培养体系。利用过表达SV40基因，我们在国际上率先建立了人精原干细胞系，该细胞系具有人类精原干细胞的体外和体内生化表型，已长期培养3.5年和大量增殖，并且，有较好的安全性。利用水凝胶和多种生长因子构建了人类原代精原干细胞的体外长期培养体系。原代人精原干细胞体外培养60天和增殖，并保持原代人精原干细胞的生化表型和特征。这将为揭示人精原干细胞分子机制和临床应用研究提供重要的细胞来源。2）揭示人精原干细胞自我更新的基因与信号转导、miRNA调控机制。发现P63基因突变导致生殖细胞凋亡及其转录调控机制，这将为男性不育的诊断和治疗提供新的靶点。SMAD3和AKT信号通路在人精原干细胞增殖中被激活。miRNA-20和miRNA-106a通过调控Stat3、Fos和Ccnd1刺激精原干细胞的自我更新。MiR-663a靶向调控NFIX、细胞周期蛋白A2、B1和E1促进人类精原干细胞自我更新并抑制其凋亡。3）构建了三维培养体系和条件培养基，诱导人类精原干细胞体外分化为有功能的成熟精子细胞，为无精症病人提供种子细胞。利用Matrigel构建三维培养体系，添加多种因子和激素建立条件培养基，分别诱导无精子症人和隐睾病人的精原干细胞体外分化为有受精和发育能力的单倍体精子细胞。4）探讨人精原干细胞分化为精子细胞的基因与非编码小RNA的调控机制，为阐释人类精子发生机制提供新的分子机理。比较人类精原细胞、粗线期精母细胞、圆形精子细胞的大规模差异基因表达谱，发现HOXs、JUN, SP1、TCF3等基因可能调控人类精原干细胞的分化。绘制正常人和非梗阻性无精子症病人精原细胞、粗线期精母细胞、圆形精子细胞的miRNA大规模差异表达谱，预测并验证这些差异miRNA的靶基因。本项目为揭示人类生殖细胞发育机制提供新的遗传与表观遗传调控机理和技术平台，对治疗男性不育、研发新型男性避孕方法都有重要意义。