精索静脉曲张不育患者精子蛋白组双向电泳技术的建立与优化 何犇 唐 .doc

精索静脉曲张不育患者精子蛋白组双向电泳技术的建立与优化 何犇 唐伟 罗华铭 郭毅 李跃华 （重庆医科大学附属第一医院泌尿外科，重庆，400016） 【摘要】目的: 建立与优化精索静脉曲张不育患者精子蛋白质组双向凝胶电泳技术。方法：采取Percoll密度梯度离心法提取精索静脉曲张不育患者精子，比较不同精子蛋白样品制备方法、不同胶条、不同上样量和不同水化方式对双向凝胶电泳结果的影响。结果：使用clean-up kit法对样品进行预处理，选取pH5-8的固相化pH梯度胶条，采用250ug的上样量和主动水化方式能获得背景干净、分辨率高的双向电泳图谱。结论：本研究成功建立了精索静脉曲张不育患者精子蛋白双向凝胶电泳平台，所获得双向电泳图谱的分辨率高、重复性好，为进一步探讨精索静脉曲张导致不育的分子机制奠定了技术基础。 【关键词】精子蛋白质 精索静脉曲张 双向电泳 蛋白质组学 中图分类号： Establishment and Optimization of Two-dimensional gel Electrophoresis for Proteomics research of sperm protein of varicocele patients He Ben, Tang Wei, Luo Huaming, Guo Yi, Li Yuehua (Department of Urology, the First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China) 【ABSTRACT】Objective: To establish and optimize a set of two-dimensional gel electrophoresis (2-DE) techniques for proteomic research of sperm protein of varicocele patients. Methods: Spermatozoa in the semen were purified by percoll gradient technique. We compared the effects of different sample preparation methods, different gels, different amount of protein loads and different rehydration methods to the 2-DE maps. Results: After the sample was treated with clean-up kit, a high resolution and clear 2-DE map for sperm protein of varicocele patients was obtained with pH5-8 gel, 250ug protein loads and initiative rehydration method. Conclusion: The 2-DE map of sperm protein of varicocele patients was successfully obtained with good repeatability and high resolution, which pave the way for the further proteomic research of infertility caused by varicocele. 【Key words】sperm protein varicocele two-dimensional gel electrophoresis proteomics Corresponding author: Tang Wei, Tel: 86-23-8901-1121。Fax: 86-23-6881-1487。E-mail：tangwei2060@126.com 随着人类基因组测序的完成，生命科学研究已进入了后基因组时代。在这个时代，生命科学的主要研究对象包括结构基因组研究和蛋白质组研究。蛋白质组(proteomics)的内涵是一个细胞、一类组织或一种生物的基因组所表达的全部蛋白质[1]，由MarcWilkins于1994年首次提出[2]，现已广泛用于生命科学的各个领域。 双向电泳（two-dimensional electrophoresis, 2-DE）是蛋白质组研究的核心分离技术之一[3]，获得分辨率高、重复性好的双向电泳图谱是蛋白质组学研究的基础。但因不