电压依赖性阴离子通道蛋白在特发性弱精子症发病机制中的作用研究

Asthenospermia is a common cause of human male infertility. However, no clear causes can be found in some cases using routine clinical examinations, which are named as idiopathic asthenospermia and are very difficult to diagnosis and treatment. In the previous study, we reported for the first time that voltage-dependent anion channels (VDAC) exist in human spermatozoa. We also found significant differences of VDAC gene expression between spermatozoa of idiopathic asthenospermia and that of normal control cases. Our results suggested that the abnormal expression or functions of VDAC maybe be concerned with the occurrence of asthenospermia. Based on previous studies, we will continue to further study the relationship between VDAC and idiopathic asthenospermia from the following aspects: detecting and comparing the expression and subcellular structure localization of VDAC protein in spermatozoa between idiopathic asthenospermia and normal control group; respectively detecting and comparing VDAC tag SNPs in peripheral blood from genetic perspective and VDAC promoter region methylation in spermatozoa from epigenetic perspective between idiopathic asthenospermia and normal control group, and analyzing genotype-phenotype correlation and the possible gene-environment interactions. Our study will systematically explore the roles of VDAC in the pathogenesis of idiopathic asthenospermia from gene and protein levels. It will be benefical to reveal new etiology of asthenospermia and to find new methods of the diagnosis and treatment for asthenospermia.

弱精子症是男性不育的主要原因之一。临床上存在大量病因不明的特发性弱精子症患者，是男性不育症诊疗的难点。前期研究中我们首次报道了人类精子中存在电压依赖性阴离子通道（VDAC）蛋白并发现特发性弱精子症患者精子中VDAC的基因表达与正常对照人群有明显差异，提示VDAC的表达或功能异常与弱精子症的发生有关。在前期工作基础上,我们将从以下方面继续深入研究VDAC与特发性弱精子症的关系：检测并比较特发性弱精子症患者与正常对照人群精子中VDAC蛋白的表达与亚细胞结构定位；分别从遗传学和表观遗传学角度检测并比较特发性弱精子症患者与正常对照人群外周血VDAC基因标签SNPs 和精子中VDAC基因启动子区甲基化状态，分析基因型-临床表型之间的关联性和可能存在的环境-基因交互作用。我们的研究从基因和蛋白水平系统探讨VDAC与特发性弱精子症发生之间的关系，有助于揭示弱精子症新的病因，发现弱精子症新的诊疗方法。

背景：VDAC2参与调控精子活力，共孵育VDAC2抗体发现精子活力参数（VSL、VCL及VAP）明显降低。研究中我们发现Clusterin与VDAC一起结合与精子表面参与精子质量的调控，与弱精子症相关。查阅文献得知Clusterin基因参与肿瘤的发生发展，我们推测clusterin可能与人精原细胞瘤的发生发展有关。目的：研究比较正常男性精子与特发性弱精子症患者精子vdac2启动子区甲基化差异，同时探讨VDAC相关蛋白Clusterin与精原细胞瘤的关系。方法：我们收集了26个正常男性和25个特发性弱精子症患者精液标本。5’-AZA处理小鼠精母细胞，qPCR验证vdac2是否受甲基化调控。Promoter Scan预测vdac2基因启动子区以及分析该启动子区CpGs，双荧光素酶报告基因验证启动子区活性。提取标本基因组DAN，经由亚硫酸氢盐行甲基化修饰测序，分析比较两组vdac2启动子甲基化程度。收集临床病例确诊为精原细胞瘤的睾丸肿瘤标本和前列腺癌行手术去势的睾丸标本，提取mRNA及蛋白，qPCR、western blot 以及免疫组织化学等方法，研究比较clusterin在睾丸精原细胞瘤中在基因和蛋白水平的表达和定位变化。结果：qPCR证明基因vdac2 mRNA水平表达较对照组明显增高（P<0.05）。双荧光素酶报告基因验证预测启动子区具有启动子活性（P<0.05）。亚硫酸氢盐修饰测序结果表明，正常人完全没有甲基化，轻度甲基化以及中度甲基化克隆数百分比分别为：83.65%±5.51%、8.73%±1.38%、7.61%±5.68%；特发特发性弱精子症患者完全没有甲基化，轻度甲基化和中度甲基化克隆数百分比分别为：76.01%±6.94%、7.14%±1.86% 16.62%±8.27%。两组比较P=0.005、P=0.02、P=0.003，有统计学意义。整体样本精子活力与甲基化程度相关性分析显示：精子活力与完全没有甲基化百分比呈明显正相关（P=0.0003），与中度甲基化呈明显负相关（P<0.001）。此外，精原细胞瘤clusterin表达明显降低。结论：vdac2启动子区甲基化程度与精子活力有显著相关性。高甲基化可导致特发性弱精子症的发生。VDAC相关蛋白Clusterin参与人精原细胞瘤的发生发展，可能与精原细胞瘤的低度恶性、对化疗和放疗的高度敏感性密切相关。