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

Establishment and optimization of two-dimensional gel electrophoresis for proteomics research of sperm protein of varicocele patients

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pH 5～8的固相化pH梯度胶条,采用250μg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(Two-dimensional electrophoresis,2-DE)图谱。结论: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2-DE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。

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.The effects of different sample preparation methods,different gels,different amount of protein loads and different rehydration methods to the 2-DE maps were compared.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 pH 5~8 gel,250 μg protein loads and initiative rehydration method.Conclusion：The 2-DE map of sperm protein of varicocele patients is successfully obtained with good repeatability and high resolution,which paves the way for the further proteomic research of infertility caused by varicocele.