应用条件基因敲除小鼠研究IGF1R对Leydig干细胞分化的影响

Which factors control the developmental process of Leydig cells? Our previous studies demonstrated that insulin-like growth factor 1 (IGF1) and LH are critical factors for Leydig cell development. In the adult whole-body IGF1 knockout testis, the Leydig cell number is only 1/6th of that in wild-type, and the knockout mouse also had lower serum testosterone，with only 1/6th of that of wild-type. At the same times, the serum LH level in IGF1 knockout mouse is also significantly reduced. Therefore, whether IGF1 regulates Leydig cell development via regulating LH secretion or directly acting on the Leydig cell itself is still unknown. In the present application, we hypothesize that IGF1 also directly stimulate Leydig cell proliferation and differentiation depending on the stage of Leydig cells. We use Cre-loxP system to bred Leydig cell conditional knockout IGF1 receptor (IGF1R) by crossing the CYP17-cre (Leydig cell specific gene) mouse with IGF1R floxed mouse. We will whether PLC and ILC proliferation is eliminated and whether the differentiation of PLC into ALC is delayed. These studies will help us to understand the regulatory mechanism of Leydig cell development and understanding the pathophysiological changes of some male hypogonadism.

是什么因子控制睾丸Leydig细胞分化？我们的前期研究表明，胰岛素样生长因子1（IGF1）和促黄体激素（LH）是Leydig细胞分化的关键因子。全身IGF1基因敲除小鼠， 成年睾丸只有1/6的正常Leydig细胞数，只合成1/6的正常睾酮量。同时，IGF1敲除小鼠也导致LH分泌显着减少。因此，IGF1影响Leydig细胞分化是通过降低LH分泌还是直接刺激Leydig细胞分化？我们认为，IGF1直接刺激Leydig细胞增殖和分化。我们应用条件基因敲除小鼠Leydig细胞的IGF1受体（IGFR）来验证IGF1是Leydig干细胞/祖细胞增殖和分化的重要因子，并且通过IGFR而起作用。这些研究的结果将有助于我们理解Leydig细胞的调节机制，也许在性腺减退、老化的男性和其它的睾酮降低的临床病例中，通过优化Leydig细胞分化以恢复产生睾酮的潜能。

在睾丸发育过程中，我们目前鉴定了与成年型Leydig细胞（ALC）的分化相关的一些细胞序列。其中最早被鉴定的是祖间质细胞（PLC），这是一种纺锤形、含LH受体和3β-羟基甾脱氢酶(3βHSD1)的细胞，它在体内能分化产生不成熟间质细胞（ILC），并最终分化成ALC。但是，对于控制Leydig细胞生长分化的因子尚未阐明。在此项目中，我们用大鼠经乙基二甲基硫砜（EDS）处理后，睾丸间质细胞开始再生，分离纯化大鼠再生的睾丸间质细胞系中的细胞，包括再生的睾丸间质祖细胞（RPLC）、不成熟细胞（RILC）和成年细胞（RALC）。RPLC、RILC和RALC分别从EDS处理后的21、28和56天的睾丸中分离。我们成功地建立了成年大鼠各阶段分离纯化Leydig细胞的技术。然后，我们创建了Leydig细胞条件敲除胰岛素样因子1受体（IGFR）小鼠（IGFRKO），比较成年野生型和IGFRKO小鼠睾丸Leydig细胞数量和睾丸Leydig细胞的成熟状态。我们通过多种试验手段了解了IGFR对睾丸Leydig细胞发育的分子机制。这些研究有助于我们了解Leydig细胞分化的调节机制，为Leydig细胞的分化提供新的调控理论基础。