DDR1基因在非梗阻性无精子症发病中调控机制的研究

Male infertility accounts for 50% of total infertility cases. Non-obstructive azoospermia is the most severe form of male infertility, manifesting no sperm in semen sample due to spermatogenesis failure in testes. The basement membrane enclosing seminiferous tubule plays an important role in the development and differentiation of male germ cells. Collagen IV constituting collagen network in basement membrane is the unique ligand of discoidin domain receptor 1 (DDR1). Our previous research showed that DDR1 expressed in seminiferous tubule. We are proposing that DDR1 is involved in the spermatogenesis failure in non-obstructive azoospermia patients. The mechanism may be that the MAPK and NF-κB signaling induced by DDR1 affect expressions and functions of matrix metalloproteinases (MMPs). The construction and dynamic of extracellular matrix and basement membrane are therefore impaired, leading to spermatogenesis failure. We are going to recruit non-obstructive azoopermia patients and control subjects, collecting testical seminiferous tubule specimen. Expression of DDR1 and MMPs will be detected in seminiferous tubule specimen. Spermatogenic cell line and Sertoli cell line will be used to study the MAPK and NF-κB signaling induced by DDR1. MMPs expression as well as cell proliferation and apoptosis will be evaluated under over expression or expression silence of DDR1. We are hoping pathogenesis of non-obstructive azoospermia could be partially elucidated in this study and providing some clues for treatment in the future.

男性不育因素在不孕症中占50%，非梗阻性无精子症（NOA）是男性不育中最严重的形式，表现为精子发生障碍而导致精液中无精子。睾丸生精小管上皮的基底膜对于生殖细胞的发育分化有重要作用，而基底膜的IV型胶原是盘状结构域受体DDR1的唯一配体。我们前期研究证实DDR1在生精小管中表达，基于此我们提出“DDR1是影响NOA精子发生障碍的调控因子”的假说，其机制可能是通过DDR1诱导的MAPK和NF-κB信号通路，影响MMPs的表达和功能，进而影响细胞外基质和生精上皮基底膜的正常构建和动态平衡，导致精子发生障碍。本研究将收集NOA患者和对照个体的生精小管，分析DDR1和MMPs的表达情况；利用生精细胞和支持细胞株研究DDR1诱导MAPK和NF-κB信号通路的机制，以及DDR1表达改变对MMPs表达和细胞增殖与凋亡的影响。本研究将有可能揭示部分NOA的发病机理，为此类疾病今后的治疗提供理论基础。

本研究主要探讨DDR1基因在非梗阻性无精子症中精子生成障碍的调控机制，分两部分进行。第一部分收集非梗阻性无精子症患者和梗阻性无精子症患者以及精液常规正常患者的生精小管标本，通过免疫组化和定量PCR研究分析DDR1，MMP2，MMP9，TIMP1和TIMP2基因的表达情况。研究结果显示，相比于梗阻性无精子症患者以及精液常规正常者，非梗阻性无精子症患者生精小管DDR1，MMP2和MMP9基因表达显著下降。第二部分是在体外细胞实验中研究DDR1通过信号转导通路调控下游基因表达情况。研究发现，在精原细胞株GC-1spg和精母细胞株GC-2spd中，DDR1基因通过ERK通路调控下游基因表达。在IV型胶原刺激下，通过DDR1调控ERK通路，下游MMP2和MMP9基因表达显著升高，ERK通路的抑制剂能够显著抑制MMP2和MMP9基因表达。在DDR1过表达实验中，MMP2和MMP9基因表达显著升高；而DDR1沉默能够显著下调MMP2和MMP9的表达。此外，DDR1沉默能够降低GC-1spg和GC-2spd细胞的增殖。本研究的研究结果提示DDR1基因在非梗阻性无精子症中精子生成障碍中可能起到一定的调控作用，将为进一步探索非梗阻性无精子症的发病机制和可能的治疗奠定基础。