基于“基因突变-蛋白质表达-多胺代谢”的非小细胞肺癌证候标记物群与中药药效物质研究

“Syndrome differentiation and treatment，formulas corresponding to syndrome” is the core concept of Traditional Chinese Medicine (TCM) in diagnosis and treatment. To elucidate deeper implication of “syndrome characteristics” of non-small cell lung cancer (NSCLC) in TCM through the studies on material basis and to study on the material basis and mechanism of TCM formula in the treatment of NSCLC have become the key issue on enriching and developing the basic theory and practical application in the treatment of NSCLC using TCM. Based on the TCM theories, the aim of the project is to establish a method to analyze the cancer gene mutation, protein expression and abnormal metabolism of polyamine related to NSCLC. To investigate the relationship among the four syndrome of “syndrome of deficiency of both qi and yin、syndrome of endogenous heat due to yin deficiency, symptom of lung and spleen qi weaken, syndrome of blood stasis with qi stagnation”, develop a biomarker group based on gene-protein-polyamine system. With the TCM formula AIDI injection and Kangai injection as the research object, study the regulating effect of two injections on syndrome biomarker group from pathologic model animals and non-small cell lung cancer patients. Using multi techniques of LC-MS-IT-TOF and UHPLC-MSn, establish in vitro and in vivo fingerprints of the prescriptions and determine the pharmacological materials spectrum. Finally, illustrate the material basis of TCM and reveal the anti-cancer mechanism of the TCM formulas through results of the pharmacokinetic and pharmacodynamic and data mining technology.

“辨证论治，方证对应”是中医药个性化诊治核心思想，如何从物质基础层面阐释非小细胞肺癌中医“证候”内涵，并以此为基础研究复方中药的抗肺癌药效物质基础与作用机制是丰富和发展中医药治疗非小细胞肺癌基础理论与应用实践的关键科学问题。本项目以中医理论为指导，围绕与非小细胞肺癌密切相关的突变基因、表达蛋白和代谢异常多胺类物质，探寻其与非小细胞肺癌“气阴两虚、阴虚内热、肺脾气虚、气滞血瘀”四类证候相关性，建立“基因-蛋白质-多胺”证候标记物群；以艾迪注射液、康艾注射液为研究对象，考察其对病证动物及临床患者体内证候标记物群调节作用，结合临床疗效确定药物对应证候与药效；采用LC-MS-IT-TOF、UHPLC-MSn手段，建立药物体外指纹图谱与方证对应体内指纹图谱，确定活性成分物质谱；对其进行药动学研究，结合药动学-药效学研究结果，运用线性回归、相关性分析等数据挖掘技术，确定中药药效物质基础，揭示作用机制。

课题通过研究机体内源性蛋白、脂类、小分子等代谢网络和复方中药代谢网络相互作用出发，建立了高灵敏度、高准确度、高覆盖率的靶标胺类亚代谢组学、靶标脂质组学、靶标甾体组学、分析方法以及肺泡灌洗液菌群、蛋白质组学、脂质组学、代谢组学的非靶标分析方法，综合分析健康与非小细胞肺癌证候机体的变化，建立非小细胞肺癌证候表征机体内源性代谢网络与证候标记物群；用于检测中医已诊断非小细胞肺癌“气阴两虚、阴虚内热、肺脾气虚和气滞血瘀”证型患者、健康志愿者血浆和尿液中内源性物质的水平，皆在筛选基于不同症候非小细胞肺癌的生物标志物群，并通过显著性分析、判别分析、ROC分析、逻辑回归分析、套索回归分析等相关性分析对标记物群进行了确证。确定CLIC1、AQR等基因，氯化物细胞内通道蛋白1等蛋白，瓜氨酸等氨基酸， LPC 18:2 sn 2等脂类，甾体激素雄烯二酮（女性患者）等非小细胞肺癌证候的标记物群。通过组织切片、生化指标、动物表观状态观察以及临床已确定的标记物群对动物模型进行确证。选取临床常用抗肺癌复方中药艾迪注射液、复方苦参注射液、康艾注射液为研究对象，分别考察其对“气阴两虚、阴虚内热、肺脾气虚和气滞血瘀”四类病证模型动物体内症候标记物群的调节作用，并以对标记物群的调节作用作为药效评价指标；同时建立上述药物体内、外指纹图谱的快速分析鉴定方法；分别对模型动物以及健康动物给予上述药物，建立方证对应药物体内物质指纹图谱；结合体内外指纹图谱确定药物活性成分物质谱，对活性成分进行药代动力学研究，确定了中药药效物质基础，以揭示作用机制。本研究为丰富中医药个性化诊治核心思想“辨证论治，方证对应”提供依据，为复方中药现代化研究提供共性分析方法与关键技术。