【作者】 魏鉴腾；

【导师】 林秀坤； 韩丽君；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋药物学， 2012， 博士

【摘要】 肿瘤是危害人类健康的重大疾病。胰腺癌是人类最常见的恶性肿瘤之一。由于诊断困难和预后效果差，导致胰腺癌病人5年的存活率仅为3-5%。因此，寻找高效低毒的新型药物和新的治疗方案成为目前研究者主要关注的问题。齐墩果酸（OA）是一种五环三萜类化合物，对多种肿瘤显示了良好的活性。近年来，OA的抗肿瘤活性由于其高效和低毒而广受研究者青睐。但其抗肿瘤作用的机制上不明确。本课题研究了OA抗胰腺癌活性以及其具体的作用机制。MTT结果显示，OA能够抑制胰腺癌细胞增殖，呈浓度依赖性关系，其IC50为46.35g ml-1。流式细胞仪检测结果发现，OA能够将胰腺癌细胞的细胞周期阻滞于S期和G2M期。AnnexinV/PI双染、heochst33342染色和基因组DNA检测发现，OA能够诱导胰腺癌细胞出现凋亡现象。进一步的研究发现，OA能够诱导胰腺癌细胞内ROS产生，从而了细胞进入线粒体凋亡通路和溶酶体凋亡通路。其次，实验研究了OA对人脐静脉内皮细胞（HUVECs）增殖、迁移和管腔形成的作用和影响。结果显示，OA处理8h，60和80g ml-1的抑制率分别为6.91%，15.34%；处理24h时，抑制率分别为11.22%，41.88%。平板划痕和Transwell小室结果显示，40g ml-1的OA组，迁移率分别为71%和77%，随着浓度增加，迁移抑制率也随之升高。管腔形成实验结果显示，OA浓度达到40和60g ml-1时，管腔形成的区域降为33%和20%。Western blot检测发现，OA能够抑制HUVECs中VEGF的表达量和分泌量。最后，实验还检测了OA联合5-FU用药对胰腺癌细胞的作用效果，结果显示，OA和-FU联合用药效果要明显高于其单独用药，能够增强对肿瘤细胞增殖的抑制和促进肿瘤细胞凋亡，具有明显的协同作用。进一步研究发现，联合用药改变了线粒体膜电位，但并没有导致cyto C的释放，因此不能启动线粒体凋亡通路；联合用药可以改变溶酶体膜的通透性，导致组织蛋白酶泄漏，从而启动溶酶体凋亡通路。尽管如此，联合用药还能导致线粒体通路相关蛋白Bcl-2、Bax和Survivin的表达，实验还发现，联合用药作用并不能改变p53的表达，但可以上调NF-κB的水平。综上所述，OA可以通过多种途径抑制肿瘤的生长和发展，并且可以联合5-FU达到协同抗肿瘤的效果，因此OA有潜力成为抗胰腺癌的一种新的药物或方案。更多还原

【Abstract】 Pancreatic cancer is one of the most common malignant tumors in man. Despiteadvances in surgical and non-surgical treatments, it remains a big challenge duo to itspoor prognosis; an overall5-year survival rate is only3-5%due to the lack ofsymptoms and screening techniques for early detection, aggressive metastaticbehavior, and resistance to conventional chemotherapy and radiotherapy regimens.Therefore, there is an urgent need to develop novel agents and alternative strategiesfor the treatment of pancreatic cancer.Oleanolic acid (OA), a pentacyclic triterpenoid, exhibits potential anti-tumoractivity against many tumor cell lines. Due to its marked antitumor effects andpharmacological safety, the antitumor activity of OA has attracted more attention.However, little is known about the underlining mechanism of OA induced anticanceractivity. In the present study, we study the anti-tumor activity of OA on pancreaticcancer cells and its potential molecular mechanism. The results showed that theproliferation of Panc-28cells was inhibited by OA in a concentration-dependentmanner, with an IC50value of46.35g ml-1, as determined by MTT assay. The cellcycle was arrested in S phase and G2/M phase by OA. The study also showed that OAcould induce remarkable apoptosis, evidenced by increased percentage of early/lateapoptotic cells, DNA ladder and nuclear morphology change. Further study revealedthat OA could induce apoptosis possibly via ROS-mediated mitochondrial andlysosomal pathway in Panc-28cells. Secondly, the study investigates the effects of theoleanolic acid on the proliferation，migration and the formation of tube-like structurein human vascular endothelial cells (HUVECs). MTT assay showed that the inhibitionrates of HUVECs treated with60and80g ml-1of oleanolic acid for24h were11.22%and41.88%respectively. Treatment of HUVECs significantly inhibited the cell migration in a dose-dependent manner. The vascular index of HUVECs treatedwith40and60g ml-1were33%and20%respectively. Western blot analysis showedthat treatment of the cells with oleanolic acid significantly attenuated the expressionand secretion of VEGF. Thirdly, the combination of OA and5-FU on the humanpancreatic Panc-28cells was studied. The results showed the combined use of OA and5-FU synergistically potentiated cell death effects on Panc-28cells, and the apoptoticeffect was also increased. Further study revealed that the combined treatment couldenhance mitochondrial depolarization, lysosomal membrane permeabilization (LMP)and leakage of cathepin D, while the release of cytochrome C did not displaysignificant changes. The expression of apoptosis related proteins was also affected incells treated with the combination of OA and5-FU, including activation of caspases-3and the expression of Bcl-2/Bax, Survivin. The result also showed that OAdid notaffect the expression of p53, and the level of NF-κB was upregulated.In conclusion, OA can inhibit tumor growth and development through a varietyof tagarts, and the use of combination with5-FU can display synergistic anti-tumoreffect. OA has a potential to become a novel agent and alternative strategy for thetreatment of pancreatic cancer.更多还原