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小鼠附睾管腔液蛋白的iTRAQ定量蛋白质组学研究
中文摘要

目的 世界卫生组织(WHO)的调查结果显示,不育症的发病率在世界范围内呈上升趋势,占已婚育龄夫妇的15%左右,其中男方因素约占50%。男性不育的病因复杂,如睾丸中精子发生异常及附睾中精子成熟紊乱都将引起男性不育。附睾管腔液蛋白在精子成熟过程中起着重要的作用,但是其蛋白成分不明,严重阻碍了精子成熟机制及男性不育致病机理的相关研究。因此,探明小鼠附睾管腔液的蛋白成分及附睾头和附睾尾的差异表达蛋白将成为本课题的研究重点。 方法 本课题首次采用同位素标记相对和绝对定量(iTRAQ)偶联2D-LC-MS/MS技术对小鼠附睾管腔液蛋白进行研究。通过分析小鼠附睾头和附睾尾部的管腔液,鉴定小鼠附睾管腔液蛋白成分,建立小鼠附睾头和附睾尾差异表达蛋白谱。利用生物信息学技术,对差异表达的蛋白进行GO分析、KEGG通路富集及STRING蛋白相互作用网络研究。采用免疫印迹及免疫荧光技术,对差异表达的蛋白进行研究,以验证蛋白质组学结果。结果 本课题首次鉴定了2132个小鼠附睾管腔液蛋白,发现395个在附睾头和附睾尾呈动态表达的蛋白,鉴定128个差异表达的蛋白,其中46个在附睾头部富集, 82个在附睾尾部富集。生物信息学分析发现,这些差异表达的蛋白主要位于细胞质、囊泡、外泌体及胞外囊泡中,具有广泛的结合能力,参与多项生理代谢过程。而Acly和Mdh2分别位于附睾头和附睾尾富集的蛋白作用网络的中心。通过对新发现的附睾管腔液蛋白Pdzkl和Spink5的免疫印迹和免疫荧光的研究,验证了本课题蛋白质组学的研究结果。 结论 iTRAQ技术是研究附睾管腔液蛋白的有力工具,相较传统的2D-PAGE技术具有极大的优势。本课题首次系统的揭示了小鼠附睾管腔液的蛋白组成,鉴定了2132个蛋白,发现128个差异表达的蛋白,并利用免疫印迹和免疫荧光技术对蛋白质组学的数据进行了验证。本课题的研究结果将为研究精子成熟的机制及男性不育的致病机理提供基础。 关键词 附睾;精子;iTRAQ;蛋白质组学;生育力

英文摘要

Objective According to the report of WHO (World Health Organization), infertility affects 10-15% of reproductive-aged couples worldwide, and half of the cases are due to male factors. Many factors contribute to male infertility, such as impaired spermatogenesis in the testes, and sperm maturational disorders in the epididymis. The luminal proteins of eoidiymis are essential for sperm maturation, but the protein compositions are still obscure, which seriously impact the studies on sperm maturation and male infertility. Thus, identifying the luminal proteins and the differentially enriched proteins between the caput and cauda is the main object of this study. Methods For the first time, isobaric tag for relative and absolute quantification (iTRAQ) coupled 2D-LC-MS/MS approach was applied to uncover proteins that were differentially enriched in the mouse epididymal fluid of caput and cauda. Biological information analysis was performed on the differentially abundant proteins through GO annotations, KEGG pathway and protein interaction. Two differentially enriched proteins were further studied by Western blotting and immunofluorescence. Result For the first time, as many as 2132 proteins were detected in the mouse epididymal fluids, 395 proteins with dynamic expression were found, and 128 differentially enriched proteins were identified, of which, 46 were caput fluid enriched and 82 were cauda enriched. GO annotations indicated that the cellular component of these proteins manly localized in the cytoplasm, membrane-bounded vesicles, extracellular exosome and extracellular membrane-bounded organelle. Molecular function and biological process of these proteins were very similar, binding activity and metabolism were the main functions enriched. Acyl and Mdh2 was the center of protein interaction networks in the caput and cauda enriched proteins, respectively. And the proteomic results were validated by Western blot and immunofluorescence. Conclusions iTRAQ is a powerful method for the study of epididymal luminal proteins, compared with tradictional 2D-PAGE. We have first identified as many as 2132 proteins in the mouse epididymal fluid, largely increased the number of proteins have been identified. These results will provide the basic information for further exploring the mechanism of sperm maturation and male infertility. Keywords Epididymis; Sperm; iTRAQ; Proteomics; Fertility

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