芫菁药用资源的抗肿瘤价值评价及其机制

The cantharidin and its derivatives orginated from Meloidae were used as clinically important anticancer drugs, but the anti-tumor effects for different species of Meloidae exsited a huge difference. Therefore, systematically study and dig this valuable medicinal insect resources is particularly important. This project will systematically identify and classify the Meloidae species from southwest of China. Moreover, the content of cantharidin and salt cantharidate of different Meloidae species will be inveestigated, and the practical Meloidae fingerprint technology will be built. Based on the selecting Cantharidin as positive standard, several cell strains such as liver cancer cell will be used for determining the activity of different Meloidae species with different regions. Selecting cantharidin and salt cantharidin as the activity indicators, some ideal of excellent geographic species will be obtained. The compounds from this excellent geographic species with anti-tumor activity will be isolated and identified. 3-4 active ingredients with significant effect will be evaluated in vivo antitumor activity experiments, and 1-2 active ingredients will be used for investigating the possible anti-tumor mechanism, such as apoptosis, cell cycle, angiogenesis, and metastatic potential. Based on the project above, establishing a scientific and valuable evaluation system of Meloidae antineoplastic will help for providing a scientific basis of better conservation and utilization this valuable insect drug resources.

芫菁科昆虫中含有的斑蝥素及其衍生物是临床上重要的抗癌药物，但不同种芫菁的抗肿瘤效果差异巨大。因此，系统整理并深入挖掘这一宝贵的昆虫药用资源显得尤为重要。本项目拟对西南地区丰富的芫菁种类进行系统的鉴定分类、归纳整理；在此基础上，测定不同芫菁中斑蝥素及斑蝥素衍生物的含量（作为初步活性指标成分），建立简单实用的芫菁指纹图谱库；以斑蝥素为阳性对照，测定不同地域不同种的芫菁对人肝癌等几种细胞株的抑制活性，初步筛选出效果理想的优良地域种，针对优良种进行大量采集，分离鉴定其新型抗肿瘤活性成分；筛选出3-4种效果显著的活性成分进行体内外综合抗肿瘤活性评价，进一步选取1-2种体内抗肿瘤效果显著的成分进行抗肿瘤机制的深入研究，从细胞凋亡、细胞周期、血管新生、转移潜能等多方面探讨其可能的抗肿瘤机制。通过上述研究，建立科学的芫菁抗肿瘤药用价值评价体系，为更好的保护和利用这一宝贵的昆虫药物资源提供科学依据。

对西南地区的芫菁进行归纳整理，采集到芫菁科昆虫4属，30余个地域种。建立了新型的提取方法，避免了斑蝥素的影响，进而对芫菁中斑蝥素外的成分进行抗肿瘤活性研究。由斑蝥素盐类组成的结合斑蝥素具有明显的抗肿瘤作用。利用气质联用仪和液相联用仪鉴定这些芫菁体内的成分，并初步建立芫菁的GC-MS和HPLC指纹图谱分析及定量方法，进一步建立了综合质量评价体系，筛选到优势地域种。.进一步分离鉴定、人工合成新型高抗肿瘤斑蝥素衍生物，并明确了这些化合物的抗肿瘤活性以及其抗肿瘤部分机制。斑蝥素、结合斑蝥素及斑蝥素衍生物均对多种癌细胞具有很好的抑制作用，能引起多数肿瘤细胞出现G2/M 期阻滞，但也有少部分的肿瘤细胞周期阻滞于G0／G1期（如对肺癌H-1299）；且多数能引起癌细胞的调亡。其中斑蝥素酸镁效果最好。斑蝥素酸镁可以显著降低裸鼠皮下移植瘤的体积和肿瘤，并可引起明显的调亡。斑蝥素酸镁处理肝癌细胞之后，共发现了298个差异表达蛋白,包括TUBA8等诸多蛋白质。这些差异表达蛋白质中，与肿瘤相关蛋白质31个，其中4个蛋白质显著上调，27个蛋白质显著下调。最显著信号转导通路为MAPK通路。进一步试验显示：斑蝥素酸镁可能是通过抑制PP2A 活性进而抑制ERK1/2通路来实现对肝癌细胞增殖的抑制作用。