基于iTRAQ技术的肾阳虚不育小鼠睾丸差异蛋白表达研究

Study on TesticularDifferential Protein Expression in Kidney-Yang Deficiency Infertile Mice Basedon iTRAQ

目的应用iTRAQ技术研究肾阳虚证小鼠睾丸的差异蛋白表达,探讨肾阳虚引起雄性不育的机制。方法选用20只6周龄雄性昆明小鼠,随机分为空白对照组及肾阳虚证组,每组10只,肾阳虚组予氢化可的松25 mg/(kg·d)腹腔注射,空白对照组予生理盐水0.2 mL/d腹腔注射10 d。提取睾丸总蛋白,利用iTRAQ技术对两者进行蛋白质组质谱检测,并通过筛选差异蛋白、GO富集以及Pathway信号通路富集等生物信息学方法分析两组间小鼠睾丸的差异蛋白表达。结果与空白对照组相比,肾阳虚组小鼠睾丸表达的差异蛋白共87个,其中27个上调,60个下调;显著富集于生物过程中"生殖"或"生殖过程"或"精子生成"的差异蛋白18个;主要集中在核糖体通路、PPAR信号通路及钙离子信号通路中信号转导分子的活性改变。GO与Pathway共富集于Rps5、Rps19、Rps28、Rpl11、Rplp1、Rplp2核糖体蛋白。结论肾阳虚证可能通过调节核糖体蛋白的表达进而影响细胞增殖、凋亡、分化及信号转导,或下调表达对精子有保护作用的分子,或影响睾丸脂质代谢及激素水平,从而引起睾丸中生精细胞的发育异常,最终导致精子活性改变及男性不育。

Objective Tostudy the differential protein expression in testis of mice with kidney-Yang deficiency by isobaric tags for relative and absolutequantitation(iTRAQ)technology,andexplore the mechanism of male infertility caused bykidney-Yang deficiency.Methods Totally 20 male KUNMINGmiceaged6 weeks were randomly divided into a control groupanda kidney-Yang deficiency group,10 mice in each.Mice in the kidney-Yang deficiency group were intraperitoneally injected with hydrocortisone25 mg/(kg·d),mice in the controlgroup were intraperitoneally injected with normal saline 0.2 mL/d.Total testicular protein was extracted 10 days later and the two groups were detected by proteomic mass spectrometryusing iTRAQ technology.The proteomic differences between the two groups were analyzed by screening differential proteins,GO enrichment,Pathway enrichment and other bioinformatics methods.Results Compared with the control group,27 up-regulated proteins and 60 down-regulated proteins were identified,a total of 87 differentially expressed proteins.Eighteen proteins were significantly enriched in biological process called‘reproduction’or‘reproductive process’or‘spermatogenesis’in the GO enrichment analyze.Changes in the activity of signal transduction molecules were mainly concentrated in the Ribosome pathway,PPAR signaling pathway and Calcium signaling pathway.Rps5,Rps19,Rps28,Rpl11,Rplp1 and Rplp2 were enriched both in GO and Pathway enrichment.Conclusion Kidney-Yang deficiency syndrome may adjust the expression of ribosomal protein,and thus affecting cell proliferation,apoptosis,differentiation and signal transduction,or down-regulating the sperm protective molecules,or affecting lipid metabolism and hormone levels in testis tocause abnormal development of spermatogenic cell,eventually leading to changes in sperm activity and male infertility.