基于凝集素芯片技术的莪术醋制增强抗肝癌效应的糖蛋白组学研究

Curcumae Rhizoma is processed with vinegar in clinical preparation and prescription. After processed with vinegar can lead drug to liver, and enhance the efficacy of treatment of hepatocellular carcinoma and improve the ability of scattered stasis and promote elimination product. These effects have validated in the clinical. However, the mechanisms are not yet clear. The previous researches revealed that the associated glycoprotein after the development of hepatocellular carcinoma showed significant differences with normal people. Based on the previous study, the project’s breakthrough point is that Curcumae Rhizoma enhances the anti-hepatoma effect. From the difference expressions of glycoprotein in hepatoma cell or tissue between Curcumae Rhizoma and processed with vinegar, the proposes of the project is to illustrate the scientific hypothesis about Curcumae Rhizoma processed with vinegar repairs their glycoprotein functions by regulating the glycoprotein expression of hepatoma cells, and enhances the effect of anti-hepatoma. The project uses new technology such as lectin microarray and UPLC-Q-TOF/MS that can high throughput and rapid identification of glycoprotein to identify the difference lectin in the occurrence and development of HCC between Curcumae Rhizoma and processed with vinegar, purify and identify the key glycoprotein. Through the biological information database, functional analysis the metabolic pathway of the glycoprotein. The project elucidates the scientific connotation about Curcumae Rhizoma processed with vinegar enhance the anti-hepatoma effection and provides a new modern research idea for traditional processing theory.

莪术为入肝经要药，其治疗肝癌等肝类疾病效应为临床所验证，临床方剂及制剂常以醋莪术入药，醋制后增强散瘀消积及抗肝癌作用，但醋制增效机制尚未明确。肿瘤的发生发展与蛋白质糖基化表达密切相关，肝细胞癌变后其相关糖蛋白表现出显著差异。基于前期研究，课题以莪术醋制增强抗肝癌效应为切入点，从生/醋莪术干预后肝癌细胞/组织中糖蛋白差异表达的视角，提出“莪术醋制通过调控肝癌细胞蛋白质糖基化表达，以修复其糖蛋白功能，进而影响细胞间识别、粘附、通信联络，从而增强其抗肝癌效应”的科学假说。课题创新性地采用高通量快速鉴定糖蛋白的凝集素芯片技术及UPLC-Q-TOF/MS等现代技术，鉴定生/醋莪术干预后与肝癌发生发展密切相关的差异表达凝集素，从中分离纯化关键糖蛋白并鉴定结构，同时利用生物信息数据库对所得糖蛋白涉及的代谢通路进行功能分析，综合阐明莪术醋制增强抗肝癌效应的科学内涵，为传统中药炮制理论现代研究提供新的思路。

《中国药典》2015年版收载莪术的两种饮片——生莪术与醋莪术；生莪术长于行气止痛，破血祛瘀；醋莪术主入肝经血分，散瘀止痛作用增强。莪术常被用于包括肝癌在内的多种癌症的临床治疗。本课题针对生、醋莪术抗肝癌机制的异同尚未明确的科学问题，对生、醋莪术抗肝癌活性进行体内外的确证，针对肿瘤组织采用凝集素芯片技术观察蛋白质糖链谱，并进行TMT标记定量蛋白质组学分析、UPLC-Q/TOF-MS非靶向代谢组学分析及蛋白组学-代谢组学两大组学的联合分析以探寻机制。.结果显示，通过体内外抗肝癌活性研究证实生、醋莪术的抗肝癌效应，且细胞、动物实验同时表明醋莪术优于生莪术，符合《药典》对莪术功效的描述：莪术临床上可用于癥瘕痞块的治疗，且“醋莪术散瘀止痛作用增强”。从肿瘤组织蛋白质糖基化的角度尝试性探索生、醋莪术抗肝癌机制的异同，结果表明，筛选所得的差异糖链或共性糖链均较少，即现有的技术手段不够充分。继而转向技术手段更为成熟的蛋白质组学和代谢组学，结果说明生、醋莪术通过抑制肿瘤细胞能量代谢相关蛋白质表达与代谢物扰动减少其能量供给，从而抑制肿瘤细胞的快速增殖，而醋莪术的抑制效果优于生莪术。通过蛋白质组学-代谢组学联合分析聚焦至对苹果酸-天冬氨酸穿梭及脂肪酸合成与降解的抑制，即生、醋莪术通过抑制上述两条通路降低肿瘤细胞的放能效率、减少供能物质脂肪酸的供给，醋莪术的作用更强。这可能是莪术抗肝癌的重要作用机制，也是经醋制后莪术抗肝癌效应增强的重要原因。.本课题将糖组学、蛋白质组学和代谢组学及其联合分析引入中药及中药炮制加工的研究领域，从整体上观察生、醋莪术抑制肝癌细胞增殖的作用机制符合中医整体观思维，继而探寻蛋白谱与代谢谱的关联性，深度挖掘生、醋莪术的药物作用靶点及代谢调控，由面及点、层层深入去探究生、醋莪术抗肝癌机制异同的科学内涵。为莪术的炮制及饮片临床应用提供科学依据，也为中药饮片与炮制的现代研究提供参考。