HIF-1α基因沉默对精索静脉曲张不育大鼠生精功能的作用及其机理研究

Varicocele (VC) is an important reason for male infertility. The important mechanism is that increased apoptosis of germ cells may be the cause of spermatogenesis failure. However, there is no efficient method to down-regulate the apoptosis of germ cells and to improve spermatogenesis. Some researches showed that testes of VC patient were under hypoxia station, and induced increase of hypoxia inducible factor-1α, which was a kind of crucial nuclear transcriptional factor, and played pro-apoptosis role in many tissues. In order to explore whether HIF-1α has regulation roles for germ cell apoptosis in testis of VC, male infertile rat models of VC will be prepared and screened, and the plasmid of HIF-1α siRNA was transfected into testis by direct injection to silence the expression of HIF-1α. Sperm densities, motivity and capabilities of fertility will be measured to analyze the effects of silencing HIF-1α for spermatogenesis of VC rats. Morphology and ultrastructure of testis will be observed, and apoptosis-related proteins will be detected to discuss the mechanism of silencing HIF-1α on apoptosis of germ cells. The goal of this study is to investigate the therapeutic potential and the possible mechanism of silencing HIF-1α in male infertility of VC. .

精索静脉曲张（VC）是男性不育的重要原因，生精细胞凋亡增多是VC致睾丸生精功能障碍造成不育的重要机制，但是尚缺乏有效降低生精细胞凋亡、改善生精功能的措施。研究表明，VC性睾丸处于缺氧状态，引起组织缺氧诱导因子-1α（HIF-1α）表达显著升高，作为一种重要的核转录调控因子，HIF-1α在多种组织细胞中发挥促凋亡调控作用。为了探索HIF-1α的表达是否对VC性生精细胞凋亡起调控作用，本课题拟构建并筛选VC不育大鼠模型，利用RNA干扰技术，通过睾丸内直接注射的转染方式沉默大鼠睾丸内HIF-1α基因表达；通过测定精子密度和活力以及生育能力，分析抑制HIF-1α基因表达对VC性不育大鼠睾丸生精功能的改善作用；通过观察睾丸组织形态和超微结构变化，测定睾丸凋亡相关蛋白的表达，探讨沉默HIF-1α基因对降低VC性不育大鼠生精细胞凋亡，改善生精功能的机理，以期为治疗VC性不育提供新的理论依据和靶点。

精索静脉曲张（VC）是男性不育的重要原因，生精细胞凋亡增多是VC致睾丸生精功能障碍造成不育的重要机制之一。研究表明，VC性睾丸处于缺氧状态，引起组织缺氧诱导因子-1α（HIF-1α）表达显著升高，作为一种重要的核转录调控因子，HIF-1α在多种组织细胞中发挥促凋亡调控作用。为了探索HIF-1α在VC不育中对生精细胞凋亡的调控作用，本课题采用传统公认的左肾静脉部分结扎的手术方式，构建并筛选VC大鼠模型，然后利用CRISPR基因编辑技术，构建HIF-1α的CRISPR-Cas9慢病毒载体，通过睾丸内直接注射的转染方式以降低VC大鼠睾丸内HIF-1α基因表达，测定精子密度和活力以及生育能力，观察睾丸组织形态和超微结构变化，并测定睾丸凋亡相关蛋白的表达。结果发现，1. 睾丸注射HIF-1α的CRISPR-Cas9慢病毒载体后，HIF-1α表达显著降低，表明HIF-1α的基因沉默效果显著；2. 与模型组相比，VC大鼠睾丸中HIF-1α表达降低后，其精子密度和活力明显改善；3. 降低HIF-1α的表达后，VC睾丸组织结构变得致密；4. HIF-1α表达降低后，睾丸生精细胞的凋亡减少，主要体现在BCL-2表达增加，BAX表达降低，caspase3表达降低。由此，我们推断，HIF-1α在VC不育中对生精细胞凋亡起着重要的调控作用。HIF-1α基因将有可能作为治疗VC性不育的靶点，从根本上改善VC致男性不育的临床难题。