背景:国内外关于邻苯二甲酸二丁酯导致雄性生殖系统畸形发生机制的探讨多集中于邻苯二甲酸二丁酯干扰胚胎期睾丸睾酮合成途径的研究,对其基因表达调控的因素及功能蛋白质之间相互干扰的网络效应却并不明了。蛋白质组学能够从整体水平反映蛋白质组分,并筛选功能相关蛋白。目的:探讨邻苯二甲酸二丁酯胚胎期暴露对胎鼠睾丸蛋白表达谱的影响,分离并鉴定差异表达蛋白质。设计、时间及地点:随机对照动物实验,实验于2006—08/2007-10在南京医科大学动物实验中心及南京医科大学生殖医学重点实验室完成。材料:将孕鼠随机分为实验组和对照组,每组5只。妊娠14~18d.方法:实验组按800mg/(kg·d)染毒孕鼠,对照组予大豆油5mL/d。妊娠21d取出胚胎大鼠睾丸,提取总蛋白,进行二维凝胶电泳分离和图像分析,筛选出的差异蛋白质点利用质谱技术进行鉴定,并选择关键蛋白进行验证。主要观察指标:①两组蛋白的电泳差异比较。②酶切,MALDI-TOF分析,数据库检索,生物信息学检索结果。③两组蛋白Westernblotting检测及免疫组织化学染色结果。结果:共筛选出33个差异表达蛋白(t≥2.831,P〈0.05),其中14个通过质谱分析和Swiss Prot蛋白数据库检索得到鉴定,包括膜联蛋白A5、过氧化物酶6、泛素羧基末端水解酶L1等。运用Western blotting,验证了膜联蛋白A5在实验组表达量明显高于对照组,通过免疫组织化学方法,发现膜联蛋白A5主要定位在胎鼠睾丸Leydig细胞中。结论:实验运用蛋白质组学方法,建立了邻苯二甲酸二丁酯孕期暴露雄性仔鼠睾丸与正常仔鼠睾丸蛋白质差异表达谱系,鉴定出14个蛋白点,确定了膜联蛋白A5在胎鼠睾丸的定位。
BACKGROUND: Mechanism studies of male reproductive system taratogenia reduced by di(nbutyl) phthalate (DBP) mainly focuses on interfering synthetic route of andrusol DBP, however, the regulatory mechanism of gene expression and mutual interference of network effects between functional protein still remain unclear. Proteome can reflect the proteins composition on a whole level, as well as screening differential proteins. OBJECTIVE: To explore the effect on fetal rat testis which in utero exposure to DBP, in addition, isolate and identify differentially expressed proteins. DESIGN, TIME AND SETTING: A randomized control animal experiment was performed at the Animal Experiment Centre and the Key Laboratory of Reproductive Medicine in Nanjing Medical University between August 2006 to October 2007. MATERIALS: A total of 10 rats with gestational age of 14-18 days, were randomly divided into the experimental and control groups, with 5 rats in each group. METHODS: The pregnant rats in the experimental group were intragastric administration 800mg/(kg.d) DBP, while 5 mL/d soybean oil in the control group. At gestational age of 21 days, rat testis was removed, extracted total protein for 2D-electrophoregram and image analysis. The screened differentially expressed protein spots were identified by mass spectrometry, and key of which was verificated. MAIN OUTCOME MEASURES: The difference comparison of electrophoresis, identification of protein by restriction enzyme, MALDI-TOF analysis, database retrieval, bioinformatics search results, as well as western blotting detection and immunohistochemistry staining were observed. RESULTS: Thirty-three differentially expressed protein spots were screened out (t≥ 2.831, P 〈 0.05), fourteen of which were identified by mass spectrometry and SwissProt protein database, consisted of Annexin A5, peroxydase-6, and ubiquitin carboxyl-terminal hydrolase L1. Western blotting showed that Annexin A5, which was mainly detected in fetal mice testis Leydig cell, had