耐多药肺结核病中医证候血清蛋白质标志物的筛选、鉴定及功能研究

China has the highest number of multidrug-resistant (MDR) tuberculosis (TB) patients in the world. In its MDR form, TB is more difficult to treat. The combination of Traditional Chinese medicine (TCM) and Western medicine therapy may play a certain role in treating MDR-TB. But, since there are no unified criteria for TCM syndrome classification, the therapeutic effects are limited. Previously, we have identified several specific proteins and established biomarkers, such as GGH, IGHG3, and HPT for TCM syndrome differentiation of TB by isobaric tags for relative and absolute quantitation combined with two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS). In the present study, we screened and identified specific proteins in the serum of patients with MDR-TB to establish the serum protein model for MDR-TB differentiation using iTRAQ-2DLC-MS/MS. In order to reveal the mechanism of the specific proteins, we detected the function of proteins. The present study will lay the scientific foundation for biological studies on the TCM syndrome differentiation of drug-resistant TB. The study has important significance in promoting the role of integrated TCM and Western medicine therapy against drug-resistant TB.

我国耐药肺结核病的发病率居世界之首。在临床上，对耐多药肺结核病（MDR-TB）的治疗尤为棘手。中医基于外邪感染和机体正虚辨证施治，治疗MDR-TB有较好的疗效。但中医辨证标准不统一，限制其治疗作用的提高。课题组前期采用iTRAQ-2DLC-MS/MS技术，发现非耐药肺结核病的证候标志性蛋白质GGH、IGHG3、HPT，为耐多药肺结核病的中医证候研究，奠定了实验依据。基于上述工作基础，本课题拟采用iTRAQ-2DLC-MS/MS技术筛选和鉴定MDR-TB中医证候血清特异性蛋白质，建立MDR-TB证候分型模型；并通过蛋白质功能研究，揭示差异蛋白质与MDR-TB中医证候的联系。本课题的研究结果对于中医防治耐多药肺结核病研究有重要意义，并为中医证候的生物学研究奠定科学基础。

我国是肺结核病(pulmonary tuberculosis, TB)负担最重的国家之一，其中耐药性肺结核病（Multidrug-Resistant tuberculosis，MDR-TB）居世界之首。MDR-TB的防治已经成为全球结核病控制工作中的关键性难题，临床迫切需要疗效显著、毒副作用小的抗MDR-TB的治疗方案。中医(Traditional Chinese medicine, TCM))兼顾机体正虚及其他脏腑的损伤，从提高机体免疫、降低药物毒副作用、减轻肺结核中毒症状等方面，联合西医抗结核药物对结核分枝杆菌的抑制作用，能提高临床治疗MDR-TB的疗效。然而，经验性的辨证方法具有一定的主观性，影响了中医的疗效。因此，研究MDR-TB证候生物标志物，辅助传统的辨证方法，规范中医辨证，将有助于提高中西结合治疗MDR-TB 的疗效。本课题纳入MDR-TB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，应用 iTRAQ-2DLC-MS/MS 技术研究MDR-TB证候的血清蛋白质组，发现β转化生长因子诱导蛋白（TGFBI）在肺阴虚证组中浓度最低（P=0.002）;枯草杆菌前蛋白转化酶（PCSK9）在阴虚火旺证组中浓度最高(P<0.0001); 趋化因子14（CCL14）在气阴两虚证组中浓度最低（P=0.004）。因此，血清蛋白质TGFBI 、PCSK9和CCL14分别是MDR-TB肺阴虚证、阴虚火旺证和气阴两虚证的潜在蛋白质标志物。并据此建立了蛋白质诊断模型，对MDR-TB中医证候分型具有良好的价值。此外，还获得了MDR-TB患者早期诊断潜在标志物: CD44抗原（CD44）、凝血因子XI（F11）、激肽原1（KNG1）、miR-4433b-5p、miR-424-5p和miR-199b-5p,并建立了MDR-TB诊断模型。本课题为MDR-TB中医证候分类提供了生物学依据，对根据不同中医证候选取不同治疗方案具有重要意义，对于提高MDR-TB的检出率具有重要意义。