基于nano LC-LTQ-Orbitrap-MS/MS的香附四物汤干预原发性痛经模型小鼠卵巢组织差异表达蛋白筛选  

Screening for differentially-expressed proteins in ovary of primary dysmenorrheal mice with Xiangfu Siwu decoction administration using nano LC-LTQ-Orbitrap-MS /MS

作　　者：刘培[1] 尚尔鑫[1] 钱大玮[1] 朱悦[1] 唐于平[1] 段金廒[1] 

机构地区：[1]南京中医药大学江苏省中药资源产业化过程协同创新中心/中药资源产业化与方剂创新药物国家地方联合工程研究中心/江苏省方剂高技术研究重点实验室,江苏南京210023

出　　处：《中国中药杂志》2016年第16期3060-3064,共5页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81573555;81202880);江苏省自然科学基金项目(BK2012456);江苏高校优势学科建设工程项目(PAPD)

摘　　要：采用nano LC-LTQ-Orbitrap-MS/MS蛋白质组学方法筛选鉴定香附四物汤干预原发性痛经模型小鼠卵巢组织中的差异表达蛋白,推测香附四物汤治疗原发性痛经的机制。采用雌激素和缩宫素联合造成原发性痛经模型,给药组连续灌胃给予香附四物汤3 d,于末次给药后1 h,取卵巢,经蛋白提取、变性、还原、烷基化、脱盐、酶解后,进行nano LC分离并在线连接电喷雾串联Orbitrap质谱分析,利用Thermo Proteome discoverer 1.4进行数据处理,筛选并鉴定差异表达蛋白,对差异表达蛋白进行生物信息学分析。随后采用Western blot法,将重要差异蛋白进行验证。正常组、模型组、给药组3组共有的差异蛋白106个,参与细胞过程的蛋白所占比例较大,其所参与的黏着连接、黏着斑等信号通路对细胞内各种信号通路均有调节作用。验证的ADRM1蛋白在模型组中,较正常组表达上调;给药后,较模型组表达下调。通过有效的比较分析,得到一些香附四物汤干预原发性痛经的差异表达蛋白,其中ADRM1蛋白有可能是香附四物汤作用的靶点。To study the mechanism of Xiangfu Siwu decoction in treating primary dysmenorrhea, differentially-expressed proteins in ovary of primary dysmenorrheal mice with Xiangfu Siwu decoction administration were screened based on proteome technology using nano LC-LTQ-Orbitrap-MS/MS. Estradiol benzoate and oxytocin were used to produce dysmenorrheal mice model. The model mice were orally administrated with Xiangfu Siwu decoction for 3 days, and 1 h after the last administration, the ovary samples were collect- ed. After protein denaturation, reduction, alkylation, desalination and enzymatic hydrolysis, identification was carried out by nano LC- LTQ-Orbitrap-MS/MS technology. The obtained data was processed by using Thermo Proteome discoverer 1.4 software. The differenti- ally-expressed proteins were screened and identified, and their biological information was also analyzed. The siznificant differentially-expressed protein was checked using Western blot technology in ovary samples. A total of 106 differentially-expressed proteins were identified during the normal, model and administration group. Most of them participate cellular processes. Adherens junction and focal adhesion pathways play a regulatory role in various cell signaling pathways. Protein ADRM1 was validated. Compared to the normal group, it was up-regulated expression in the model group. After administration, the expression of ADRM1 was down-regulated. Through the comparative analysis, a series of differentially-expressed proteins involved in primary dysmenorrheal mice with Xiangfu Si- wu decoction administration were obtained. Protein ADRM1 may become a target for Xiangfu Siwu decoction.

关 键 词：香附四物汤 原发性痛经 蛋白质组学 差异表达蛋白 

分 类 号：R285.5[医药卫生—中药学] R-332[医药卫生—中医学]