人精子表面电压依赖性阴离子通道的基础和临床研究

项目名称： 人精子表面电压依赖性阴离子通道的基础和临床研究

项目编号： No.30872575

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 王增军

作者单位： 南京医科大学

项目金额： 30万元

中文摘要： 本研究运用基因克隆和原核表达技术体外表达出VDAC重组蛋白，为研究VDAC与人类精子表面其他蛋白之间的相互作用提供了基础；运用蛋白印迹法及免疫荧光法发现VDAC蛋白在人类睾丸生精过程中合成，以膜蛋白形式存在于成熟精子的鞭毛和颈部，系统阐述了VDAC蛋白在人类精子中的存在、表达和定位特点，为进一步的功能学研究提供了依据。将特异性的VDAC抗体与活力正常的成熟人类精子体外共孵育培养，运用流式细胞技术检测出精子胞浆中Ca2+浓度降低，提示精子膜中的VDAC蛋白参与或介导了顶体反应中Ca2+的跨膜转运和交换，初步探讨了VDAC在人类精子中的生物学功能。运用实时荧光定量PCR法检测不同活力人类精子中VDAC的信使核糖核酸（mRNA）的表达，发现活力低下精子中VDAC2的mRNA表达丰度明显比活力正常精子为高，而VDAC1和VDAC3 的mRNA表达丰度在不同活力精子中没有明显差异，揭示了VDAC基因转录和蛋白表达过程中的异常可能是某些伴有弱精子症的男性不育患者的病因之一，体现了对人类精子中VDAC进行基础研究的临床和实用价值。

中文关键词： 电压依赖性阴离子通道；精子；钙离子；核糖核酸；男性不育

英文摘要： We produced recombinant voltage-dependent anion channels (VDAC) protein through gene cloning and prokaryotic expression， which provided the foundation to research the interaction between VDAC and other proteins in the surface of human spermatozoa. We found that VDAC protein was synthesized during spermatogenesis and eventually localized to the flagellum and neck of mature human spermatozoa using western blot and immunofluorescence. Our study systematically explained the presence, expression and localization of VDAC in human mature spermatozoa for further functional researc. Then mature human spermatozoa with normal motility were co-incubated and cultured with anti-VDAC antibody in vitro. The coincubation led to decreased cytoplastic Ca2+ concentration via flow cytometer, which suggested that VDAC played a role in Ca2+ transmembrane transport and exchange during acrosomal reaction. Our results investigated the biological function of VDAC in human spermatozoa. Lastly, Real-Time PCR was performed to detect VDAC mRNA abundance of human spermatozoa with different motility. We found that VDAC2 mRNA abundance in low motile spermatozoa was significantly higher than that in high motile spermatozoa. No significant differences of VDAC1 and VDAC3 mRNA abundances were found between different sperm groups. Our data elucidated that the abnormal gene transcription and protein expression of VDAC might be one of the causes of some male infertility with asthenozoospermia, whichh reflected the clinical and practical value of studying VDAC in human spermatozoa.

英文关键词： voltage-dependent anion channels; sperm; calcium ion; messenger ribonucleic acid; male infertility