【作者】 文怡；

【导师】 魏绍斌；

【作者基本信息】 成都中医药大学 ， 中医妇科学， 2013， 博士

【摘要】 研究背景：子宫内膜异位症(Endometriosis,EMT)是育龄妇女的多发病,严重影响女性生殖健康和生活质量。证候是疾病一定阶段病因、病位、病性、病势的综合表现,也是中医药理论研究的核心内容,开展中医“证候”本质研究,揭示证候的科学内涵是中医基础理论现代化进程中的关键环节,而证候生物学基础研究的突破则是证候科学内涵得以阐明的保障。血瘀证是中医临床多种疾病的基本证型,也是子宫内膜异位症的核心证候类型。在前期研究中,曾发现该证型的异位和在位内膜在组织形态学和细胞凋亡相关因子的表达存在差异,为从蛋白组学水平探索EMT的不同证候的特异性标志物的研究工作奠定了基础。对子宫内膜异位症血瘀证进行深入、系统的现代研究,有助于对本病的准确诊断和有效治疗。目的：①通过蛋白组学技术分离并鉴定EMT血瘀证、气滞血瘀证、肾虚血瘀证患者在位和异位内膜的差异蛋白质,寻找EMT证候特异性蛋白质；②从蛋白质水平探讨EMT血瘀证、气滞血瘀证、肾虚血瘀证的实质,揭示EMT血瘀证辨证的分子生物学基础,建立EMT的蛋白质分子微观辨证和诊断体系,探讨EMT证候的演变规律、证候的辨治靶点,寻找有意义的分子标记物及基因干预的靶点。方法：取6例EMTI血瘀证、气滞血瘀证、肾虚血瘀证患者增生期在位和异位内膜组织和6例增生期正常子宫内膜组织标本,将各种证型的每两例标本进行随机等量混匀,得到3组标本并提取组织总蛋白。通过双向电泳技术得到3组双向电泳凝胶图谱。检测出的表达差异点经基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-TOF/MS)及蛋白质数据库检索进行蛋白质鉴定。结果：在3组差异蛋白中进行比较,最终得到三次重复试验所获得的各证型的差异蛋白。其中血瘀证在位内膜差异蛋白2个,异位内膜差异蛋白1个。肾虚血瘀证在位内膜差异蛋白3个,异位内膜差异蛋白3个。气滞血瘀证瘀型在位内膜差异蛋白3个,异位内膜差异蛋白4个。根据各证型差异蛋白比较,可以发现膜联蛋白在肾虚血瘀证在位内膜和异位内膜均有高表达,肌球蛋白轻链3在气滞血瘀证在位内膜和异位内膜均有高表达。结论：EMT血瘀证、气滞血瘀证、肾虚血瘀证之间蛋白质组成分存在差异,这些差异可能是证候实质的内在根源。这些差异蛋白可能成为EMT不同证候发病的候选生物标志物。更多还原

【Abstract】 Background:Endometriosis (EMT) is a frequently-occurring disease in women of childbearing age. It imposes a dramatic impact on women reproductive health and quality of life. Syndrome is the comprehensive performance of etiology, location, quality and trend of diseases in a certain phase, and it’s the central conception of theoretic research of traditional Chinese Medicine (TCM). Therefore, to investigate the innate character and to reveal the scientific essence of syndromes are the crucial point of modernization of TCM theories. Furthermore, the breakthrough of biological research about syndromes might confirm the clarification of its scientific essence. Blood stasis syndrome is a common pattern in various clinic diseases and is the key pattern in EMT. In previous research, the differences on histological morphology and apoptosis associated factors between ectopic and reigning endometrium were revealed, that applied a foundation for further study on specific markers among EMT syndromes by proteomics. It is predominantly useful for EMT’s accurate diagnosis and effective management when the profound and systemic study on syndrome of blood stasis of EMT is accomplished.Objective:①To separate and identify the global protein by proteomics from EMT ectopic and reigning endometrium in3syndromes including blood stasis syndrome, qi stagnation and blood stasis syndrome as well as kidney deficiency and blood stasis syndrome, and to reveal the specific protein for EMT syndrome.②To detect the innate characters of the three syndromes of EMT, to investigate the molecular biological basis for syndrome differentiation of blood stasis of EMT, to establish the system of microscopic identification and diagnosis for EMT protein, to discover the transmission principles of EMT syndromes and their targets, and to search the meaningful molecular marker and genetic associated targets. Methods:Tissues from ectopic and reigning endometrium in proliferative phase in EMT patients were extracted, including6of blood stasis syndrome,6of qi stagnation and blood stasis syndrome, as well as6of kidney deficiency and blood stasis syndrome, and6specimens of normal endometrium in proliferative phase were extracted. Then each two specimens in all syndromes were randomly and isometricly mixed, consequently3groups of specimens were achieved to extract the total protein. Finally3groups’spectrums of running gel were accomplished by two-dimensional electrophoresis, and the different points were measured by MALDI-TOF-TOF/MS and protein database.Results:In blood stasis syndrome, two differential proteins from reigning endomutrium were obtained, while one from ectopic endometrium was obtained. In kidney deficiency and blood stasis syndrome, three differential proteins from reigning endomutrium were obtained, while three from ectopic endometrium were obtained. In qi stagnation and blood stasis syndrome, three differential proteins from reigning endomutrium were obtained, while four from ectopic endometrium were obtained. The annexin was highly expressed in both reigning and ectopic endometrium of kidney deficiency and blood stasis syndrome. The myoglobulin light chain3was highly expressed in both reigning and ectopic endometrium of qi stagnation and blood stasis syndrome.Conclusion:There are differential protein expressions among EMT three syndromes, which might be the inner origin of syndrome characters, and these differential proteins might be as the candidate biomarkers for pathogenesis of EMT various syndromes.更多还原