人睾丸间质干细胞增殖和分化的调节机制研究

Human stem Leydig cells (SLC) can be used to treat male hypogonadism. SLC has two characteristics: self-renewal and differentiation into Leydig cell lineage. However, the controlling mechanism for SLC proliferation and differentiation is unclear. Our recent research supports that SLC exists in the outer layer of the human seminiferous tubule and can be induced to differentiate into Leydig cell lineage, and that the seminiferous tubule provides with the supporting forces. The goals in this application are: 1) to establish a novel in vitro culture system using Leydig cell-free human seminiferous tubules and to determine the mechanism of growth factors and hormone (platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I and luteinizing hormone)that control SLC self-renewal and differentiation; 2) to set up seminiferous tubule culture and induce the differentiated Leydig cells, and isolate human progenitor, immature and adult Leydig cells to determine the mechanism and the regulation of proliferation and differentiation; and 3) to purify SLC and bone marrow mesenchymal stem cell and compare them to progenitor, immature and adult Leydig cells by microarray of whole genomics in order to find the pathways of regulating the proliferation and differentiation.

人睾丸间质干细胞（SLC）可用于治疗男性性腺机能减退。SLC具有自我更新和向睾丸间质细胞谱系分化的能力。但是，调控SLC增殖和分化的机制尚不清楚。我们最新数据支持，人SLC存在于曲细精管外层，其提供SLC支承，并可以被诱导增殖和分化。本项目的研究目标是：1）建立崭新的人睾丸曲细精管体外培养系统，为研究人SLC的增殖和分化提供新型实验手段，进一步以此系统了解外源性生长因子和激素（血小板衍生生长因子、成纤维生长因子、胰岛素样生长因子-I和促黄体生成激素）调节SLC增殖和分化的机制；2）从人体外曲细精管诱导分化睾丸间质细胞，以建立纯化人睾丸间质细胞谱系细胞（祖细胞、未成熟细胞和成年细胞）的方法，从而分析它们的增殖和分化的调控机制；3）建立纯化人SLC和骨髓干细胞的方法，纯化这些干细胞并进行基因芯片分析,与祖细胞、未成熟细胞和成年细胞的基因表达进行比较，找到增殖和分化的调节通路。

睾丸间质干细胞（SLC）可分化为睾丸间质祖细胞（PLC）、幼细胞（ILC），最后发育成为成年的睾丸间质细胞（ALC）。然而，其发育过程中确切的调节机制尚不清楚。我们假设，睾丸间质干细胞和幼细胞的发育受到局部生长因子的影响。基因芯片显示激活素I型受体（Acvr1）和激活素A受体II型样1（Acvrl1）在睾丸间质细胞谱系中的表达水平是这样的：睾丸间质干细胞>幼细胞=未成熟=成熟睾丸间质细胞。这表明他们的配体激活素可能在睾丸间质干细胞和幼细胞的增殖和分化中发挥重要作用。当曲细精管与1或10ng / mL激活素A共培养3天，可浓度依赖性的增加EdU与睾丸间质干细胞中结合；当睾丸间质幼细胞与1或10 ng / mL激活素A培养2天，可浓度依赖性的增加3 H-胸苷与睾丸间质幼细胞的结合，高达200％。实时PCR分析显示激活素A主要增加Pcna的表达，但减少Star，Hsd3b1和Cyp17a1的表达水平。此外，激活素A还显著抑制基底和促黄体激素刺激的雄激素的生成。总之，激活素A主要刺激睾丸间质干细胞和幼细胞的增殖，但抑制睾丸间质干细胞和幼细胞分化进入大鼠睾丸间质细胞谱系。