基于蛋白质组学方法的“癌毒”病机研究

Cancer toxin theory is a kind of innovation of TCM by Professor Zhou Zhongying according to years of clinical experience.Previous studies have summarized the experience and diagnostic methods of differentiation tumor from "cancer toxin ",demonstrated that cancerous toxin exists objectively in tumor clinical syndrome,tested the remarkable curative effect of anti-cancer prescription under the guidance of Cancer toxin. Previous studies highlight the important position of Cancer toxin in cancerous tumors and in development, in need of deeply systematic research..Based on previous studies, the project follows traditional Chinese medicine's (TCM's) own characteristics,takes iTRAQ and MALDI-TOF-MS as technical supports, takes effective prescription as intervention, takes tumor tissue,cell and peripheral blood as objects, overall researchs the Protein expression spectrum in the intervention of Cancer toxin and effective prescription，analysis the relationship of Cancer toxin and dynamic changes Protein components, expression level and modified state from the overall Angle.To expression differences apparent function protein, We adopt PCR quantitative and Western blot validation. Through the overall characterization of Protein microarray and accurate analysis of key proteins, we obtain the function protein network and biomarker closely related Cancer toxin.Establish Cancer toxin -Protein spectrum database,explore the essence of cancerous toxin from the perspective of protein control network at the molecular level, expound scientific connotation of Cancerous Toxin Theory, initiate new ways to study TCM pathogenesis, and provide important new ideas for system research of complicated TCM theories.

癌毒理论是国医大师周仲瑛据多年临床提出的创新性中医理论，前期研究总结了从癌毒辨治肿瘤的经验及诊疗方法，证实了癌毒在肿瘤临床证候中的客观存在，验证了癌毒病机指导下形成的抗癌方疗效，凸显出癌毒在肿瘤发病中的重要地位，急需对其进行深入系统研究。项目以前期研究为基础，遵循中医学自身特点，以iTRAQ标记串联质谱技术为支撑，以有效方为干预手段，以肿瘤组织、细胞及外周血为对象,全景式研究机体在癌毒及有效方干预下蛋白质表达谱，从整体的角度分析癌毒与动态变化的蛋白质组成成分、表达水平及修饰状态的关系；对表达差异明显的功能蛋白PCR定量及Western blot验证；通过蛋白芯片整体表征和重点蛋白精确分析获得与癌毒密切相关的功能蛋白网络及标志物，建立癌毒-蛋白质谱数据库,在整体及分子水平从蛋白质调控网络探讨癌毒本质，诠释癌毒理论科学内涵，开创中医病机研究新途径，为中医复杂理论体系系统研究提供重要的新思路。

本课题以前期研究为基础，运用蛋白质组学技术，通过体内复制H22小鼠肿瘤模型，体外选用人肝癌SMMC-7721细胞株进行实验，用光镜、电镜进行形态学观察，TUNEL法检测凋亡率, Elisa法检测端粒酶浓度，以TMT标记结合LC-MS/MS法，观察消癌解毒方干预后，各组肿瘤组织、细胞及外周血差异蛋白的表达情况，应用GO分析和IPA软件分析，找出消癌解毒方抗癌机制相关的下游生物标志物。从整体的角度分析癌毒与动态变化的蛋白质组成成分、表达水平及修饰状态的关系，实时定量PCR法检测差异表达基因，Western blot法验证差异表达蛋白，同时运用蛋白芯片技术，观察消癌解毒方干预后人肝癌SMMC-7721细胞趋化因子蛋白表达的影响。..研究结果表明：消癌解毒方对荷瘤动物模型的抑瘤作用明显，同时对体外培养的肿瘤细胞也有明显的抑制作用。蛋白质组学结果鉴定出多种差异蛋白，差异表达蛋白涉及细胞凋亡、细胞增殖、细胞周期、癌症、脂质代谢、肿瘤生长、免疫等多个方面。还能调控 LXR/RXR活化通路、补体系统、细胞增殖、细胞凋亡、肿瘤生长等多条细胞信号转导通路。实时定量PCR验证结果显示消癌解毒方对凋亡相关基因bcl-2、bax、caspase-3 等mRNA表达有影响，Western blot法检测显示对凋亡相关蛋白Bcl-2、Bax、Caspase-3等，增殖相关蛋白 Ras、Raf、Mek、Mapk等,热休克蛋白相关HSP27、HSP60、HSP70及细胞周期相关Cyclin-D1、P21、P27等蛋白表达均有影响。..本研究为消癌解毒方的临床应用提供微观实验依据，同时也为进一步探讨消癌解毒方的分子机制，丰富中医“癌毒”理论的内涵打下基础。