基于Label-free定量技术及异病同证理论的妇科实寒证患者血浆蛋白质组学研究

Study on Plasma Proteomics of Gynecological Patients of Excess Cold Syndrome Based on Label-free Quantitative Technology and Theory of Same Syndrome in Different Diseases

目的基于Label-free定量蛋白质组学及异病同证理论探究妇科实寒证的生物学基础。方法纳入原发性痛经实寒证患者、子宫内膜异位症实寒证患者及健康女性各8例,分别为痛经组、内异症组、正常组。采集各组月经周期第2日的血浆样本进行蛋白提取、胰酶酶解和超高效液相色谱-质谱联用分析后得到原始数据,依据筛选标准获得差异表达蛋白,并进行交集映射,获取共有的实寒证差异表达蛋白,进行生物信息学分析。结果痛经组与正常组共筛选出219个差异表达蛋白,内异症组与正常组共筛选出180个差异表达蛋白。经交集映射得出共有的实寒证差异表达蛋白97个,其中46个蛋白上调、51个蛋白下调。对差异表达蛋白进行功能分类统计主要涉及细胞过程、对刺激的反应、代谢过程、免疫过程、生物黏附、抗氧化活性、细胞运动等。GO富集分析显示:上调蛋白主要集中在脂质转运、炎性反应、细胞迁移、细胞运动、细胞间黏附等生物学过程;下调蛋白主要集中在细胞黏附、细胞连接等生物学过程。蛋白结构域富集分析和聚类分析显示:上调蛋白主要涉及免疫球蛋白结构域;下调蛋白主要涉及亲环蛋白型肽基-脯氨酰顺反异构酶/CLD、14-3-3蛋白等结构域。KEGG通路富集分析和聚类分析结果显示:上调蛋白主要参与Th17细胞分化、谷胱甘肽代谢等通路;下调蛋白主要参与Hippo信号通路、局灶性粘连、白细胞跨内皮迁移等通路。蛋白相互作用网络中筛选出VCL、ACTR2、ACTN1等6个核心蛋白。结论妇科实寒证发生机制主要涉及代谢、免疫调节、炎性反应、氧化损伤、内皮细胞功能受损等生物学过程和信号通路。利用蛋白质组学获得的差异表达蛋白及其涉及的信号通路对揭示妇科实寒证的生物学基础有重要意义。

Objective To explore the biological basis of gynecological excess cold syndrome based on Label-free quantitative proteomics and the theory of same syndrome in different diseases.Methods Primary dysmenorrhea patients with excess cold syndrome,endometriosis patients with excess cold syndrome,and healthy women were included,who were divided into dysmenorrhea group,endometriosis group,and normal group,with 8 cases in each group.The plasma on the second day of the menstrual cycle was collected for protein extraction,trypsin digestion and ultra performance liquid chromatography-mass spectrometry analysis to obtain the original data.Differentially expressed proteins were obtained according to the screening criteria,and intersection mapping was performed to obtain shared differentially expressed proteins of excess cold syndrome.Bioinformatics analysis was performed.Results Totally 219 differentially expressed proteins were screened out in dysmenorrhea group and normal group,and 180 differentially expressed proteins were screened out in endometriosis group and normal group.After the intersection mapping,97 shared differentially expressed proteins of excess cold syndrome were obtained,of which 46 proteins were up-regulated and 51 proteins were down-regulated.The functional classification of differentially expressed proteins mainly involved cellular process,response to stimulus,metabolic process,immune process,biological adhesion,antioxidant activity,cell movement,etc.GO enrichment analysis showed that up-regulated proteins were mainly concentrated in biological processes such as lipid transport,inflammatory response,cell migration,cell movement and intercellular adhesion,and down-regulated proteins were mainly concentrated in biological processes such as cell adhesion and cell junction.Protein domain enrichment analysis and clustering analysis showed that the up-regulated proteins were mainly related to immunoglobulin domain,while the down-regulated proteins were mainly related to cyclophilin type peptidyl-prolyl cis-trans isomerase/CLD and 14-3-3 protein.KEGG pathway enrichment analysis and clustering analysis showed that up-regulated proteins were mainly involved in Th17 cell differentiation and glutathione metabolism,and down-regulated proteins were mainly involved in Hippo signaling pathway,focal adhesion,leukocyte transendothelial migration.Six core proteins including VCL,ACTR2,and ACTN1 and other were screened out in the protein-protein interaction network.Conclusion The pathogenesis of gynecological excess cold syndrome mainly involves metabolism,immune regulation,inflammatory response,oxidative damage,endothelial cell dysfunction and other biological processes and signaling pathways.Using proteomics to find differentially expressed proteins and their related signaling pathways are of great significance to reveal the biological basis of gynecological excess cold syndrome.