基于iTRAQ-2DLC-MS/MS的肺痨证候标志性血清蛋白质筛选与鉴定

China has the second largest number of tuberculosis (TB) cases in the world and ranked number one in terms of drug-resistant TB. It is difficult to treat TB with Western Medicine. Chinese Medicine has a certain effect on the drug-resistant TB, and in the diagnosis and treatment of patients with weak immunity and mycobacterium infection. Since there are no unified classification criteria for Traditional Chinese Medicine (TCM) syndrome and no standardized treatment programs, the resulting low efficacy of Chinese medicine is unable to play a role in the treatment of TB. A number of studies have found differentially expressed proteins in TB using SELDI-TOF-MS methods and established markers for TB syndrome by proteomic fingerprinting of serum. However, the technology can only detect small amounts of protein, and unable to identify and quantify differentially expressed proteins. Therefore, in the present study we used isobaric tags for relative and absolute quantification (iTRAQ) method coupled with two dimensional liquid chromatography/tandem mass spectrometric (2DLC-MS/MS) method for detecting isotope labeling of serum proteins in TB syndrome. Using 2DLC-MS/MS separation analysis, serum protein pattern model was established for the accurate quantification and direct identification of differentially expressed proteins in TB syndrome. The present study, using quantitative proteomics technology, will help to study the types TB syndrome, standardize diagnosis and treatment of TB in Chinese medicine, improve efficacy of Chinese medicine, promote the role of Chinese medicine against TB and lay the scientific basis to clarify the biological nature of TCM syndrome in TB.

我国肺痨发生率居世界第二位，耐药肺痨居世界首位，西医治疗非常棘手。中医基于患者机体正虚和痨虫感染辨证施治，对肺痨治疗具有一定疗效。然而，由于中医证候辨证标准不统一，治法治则不规范，导致疗效低，未能发挥中医对肺痨的治疗作用。课题组曾用SELDI-TOF-MS法研究肺痨证候，发现了多个差异蛋白，建立了肺痨证候血清蛋白质指纹图谱。但该技术只能捕获少量蛋白质，无法鉴定证候差异蛋白。为此，本课题应用同位素标记相对和绝对定量（iTRAQ）法结合二维液相色谱串联质谱（2DLC-MS/MS）研究肺痨证候，对血清蛋白质进行同位素标记，经2DLC-MS/MS分离，直接鉴定证候标志性蛋白质，建立肺痨证候分型血清蛋白质图谱模型。本课题应用定量蛋白质组学技术，研究肺痨的证候分型，有助于规范肺痨中医治疗的辨证施治，提高中医疗效，推动中医防痨抗痨作用，并为阐明中医证候的生物学本质奠定科学依据。

临床上肺结核病的治疗以西医抗结核药物治疗为主，由于治疗时间长，易产生药物耐受、肝损伤、药物过敏等副作用。应用中医药物进行辅助治疗，可达到良好的减毒增效作用。中医治疗的前提是辨证，基于患者机体正虚、痨虫感染程度和脏腑受累程度，进行辨证。但是，由于中医证候辨证标准不统一，治法治则不规范，导致中医对肺痨的治疗效果低。本课题纳入初治肺结核病患者，通过中医辨证分为肺阴虚证、阴虚火旺证、气阴两虚证，采用iTRAQ-2DLC-MS/MS蛋白质组学技术研究证候差异蛋白质图谱。获得了差异蛋白质γ-谷氨酰水解酶（GGH）、IgG3恒定区（IGHG3）、触珠蛋白（HPT）分别是肺阴虚证、阴虚火旺证、气阴两虚证的特异性蛋白质。通过SELD-TOF-MS-MS技术和生物信息学方法，建立了4679.7、3961.7、8891.2、5646.4、9416.7 m/z 蛋白峰构建的肺结核中医证候诊断模型；该诊断模型对肺阴虚、阴虚火旺证、气阴两虚证具有良好的诊断价值。经临床病理分析，发现了肺结核中医证候与临床病理的相关性，即肺阴虚证以结核结节等增生性病变为主；阴虚火旺证以空洞、干酪样坏死等变质性病变为主；气阴两虚证以重症肺结核病为主。并发现了肺结核中医证候在病程中的演变规律，即：证候从肺阴虚证→阴虚火旺证→气阴两虚证转归，临近死亡为阴阳两虚证；治疗后患者的证型从气阴两虚证→阴虚火旺证→肺阴虚证转归。早期、轻度肺结核病为肺阴虚证，重症、晚期肺结核病为气阴两虚证。此外，本课题还获得了肺结核病早期诊断标志性蛋白质胞外超氧化物歧化酶（SOD3）、蛋白S100-A9（S100A9）和基质金属蛋白酶9（MMP9），在肺结核病患者中表达与正常人、肺炎、肺癌患者均有显著差异，并建立了早期诊断模型。获得了肺结核病理变化的蛋白质标志物补体C4b、纤连蛋白（FN）和脯氨酸肽酶（PEPD）。通过本实验研究，建立iTRAQ-2DLC-MS/MS筛选-GO/KEGG分析- Western blot-ELISA技术平台，优化了肺结核中医证候学研究技术，服务临床肺结核中医证候研究。本课题对于完善肺结核辨证规范和辨证方法，指导临床肺结核证候分型和评价具有重要意义。