中国科学院昆明动物研究所机构知识库

大脑是人体最核心的器官，是承担语言、记忆、思维等基础性功能的物质基础，直接关乎人的生活与生存。大脑发生损伤或疾病将引起严重的后果甚至使人死亡。脑部肿瘤对大脑有致命性的伤害，也是人类健康的一大威胁，其中胶质瘤是最常见的颅内原发性脑肿瘤之一。约一半的胶质瘤为胶质母细胞瘤，胶质母细胞瘤是恶性程度很高的胶质瘤，具有治愈率低、复发率高、预后差、生存期极其有限等特点，是危害人类生命健康的恶性肿瘤之一，被列入美国TCGA (The Cancer Genome Atlas)计划选定的三种优先测定基因组的肿瘤之一。虽然临床中对胶质瘤的治疗已尽最大努力，但是收效甚微，胶质瘤患者的生存期限和生存质量依然不容乐观。因此，寻找新的、有效的抗肿瘤化合物将是十分重要和必要的。植物天然产物是药物研发的巨大宝库，从天然产物或其衍生物中分离获取的许多有各类生物活性的药物或先导化合物在生物医学领域已获得广泛应用。尤其是一些来源于植物次生代谢产物的抗肿瘤化合物表现出的极具潜力的抗癌活性，这为进一步从植物中寻找有明显抗癌药理作用的化合物奠定了理论基础和研发方向。吲哚碱是其中一类广受关注的化合物，具有非常丰富多样的结构和极具潜力的药理活性，此前发现的一些吲哚碱如长春碱、长春新碱等已经商品化，并在世界范围内应用于多种癌症的临床治疗。虽然现有的此类天然产物对胶质瘤的作用收效甚微，但以吲哚类化合物为线索，寻找与长春碱等抗癌化合物类似、结构更简单、构效关系更优化的吲哚碱化合物将会是今后抗肿瘤药物研发的一个重要的突破口。在研究团队长期的、持续的从事抗肿瘤化合物的研发中，筛选到了一个吲哚碱化合物3a-acetonyltabersonine具有较好的抗肿瘤活性，在肺癌、乳腺癌等多种肿瘤细胞系中均展现良好的抗肿瘤效果，但缺乏在胶质瘤中的研究。因此，本论文围绕3a-acetonyltabersonine在胶质瘤中的药理作用和作用机制展开研究。分离自Melodinus suaveolens的3a-acetonyltabersonine被发现在较低浓度下对胶质瘤干细胞和胶质瘤细胞系有较显著的细胞毒性，并且3a-acetonyltabersonine可以抑制胶质瘤干细胞的增殖并诱导其凋亡，这在其它的吲哚类化合物中几乎未见报道。因此3a-acetonyltabersonine具有明显的优势和研究价值。通过分析基于转录组测序的生物信息发现3a-acetonyltabersonine的作用效果与MAPK信号通路的激活及磷酸化过程相关，我们通过western blotting 证实了以上结果。伴随3a-acetonyltabersonine的作用，γ-H2AX和ATM的表达上调以及彗星电泳呈现的典型特征，揭示了3a-acetonyltabersonine的作用与DNA损伤相关。进一步的研究发现，3a-acetonyltabersonine并不对DNA造成直接的损伤，而是通过抑制DNA损伤修复来发挥其作用的。即3a-acetonyltabersonine的作用机制初步可以归纳如下：3a-acetonyltabersonine抑制了DNA 损伤的修复能力，使得DNA损伤在短时间内大量积累并激活MAPK信号通路，最终引发细胞凋亡。为进一步考察3a-acetonyltabersonine对胶质瘤的抗肿瘤效应在活体内的效果我们在小鼠模型上进行了实验。通过慢病毒诱导小鼠成瘤，病理分析确认小鼠模型为胶质瘤模型，体现着显著的胶质瘤特征。经给药实验发现，给药小鼠的生存期明显延长；免疫组化的结果也显示，经3a-acetonyltabersonine处理的小鼠，其大脑切片中凋亡细胞显著增加。以上这些证据证明了3a-acetonyltabersonine在体内也有较显著的抗胶质瘤效果。 

Brain is the most essential organ of human body and the material basis of language, memory and thinking, which is the most basic function of human beings and directly related to human life and survival. If damage or disease occurred in brain, it can couse serious results, even death. Brain tumors are fatal to the brain and a major threat to human health, of which glioma is one of the most common and aggressive intracranial primary brain tumors. About half of the glioma is glioblastoma, which is a highly malignant type with lower cure rate, higher recurrence rate, poor prognosis and limited survival time. Glioblastoma was ranked into the top 3 tumors studied by American “The Cancer Genome Atlas” (TCGA) with higher priorities, as glioblastoma is one of the malignant tumors threaten human life and health. Although the clinical treatment had been tried best, it had little effect. The survival time and quality of life of glioma patients is far from encourage. Therefore, it is important and necessary to find new and effective antitumor compounds. Plant natural products are a great valuable resource for drug development. A large number of drugs or lead compounds with biological pharmacological activity were isolated from natural products or their derivatives and they had been widely applicated in the field of biomedical. Especially, the antitumor compounds from the plant secondary metabolites showed promising activity, which lays a theoretical foundation for further reseach and an exact direction for seeking new compounds with significant pharmacological effects. Indole alkaloids is one kind of focused compound, due to their diverse structure and potential pharmacological activities. Some indole alkaloids, such asvinblastineand vincristine, had been used as best-known anticancer drugs in worldwide for various cancers in clinical treatment. Although those natural products just exerted little effect on glioma, take indole compounds as the basis and search for vinblastine analogs with simple structure andoptimized structure-activity relationship will be an significant breakthrough in the development of anticancer drugs.Our research team were engaged in developing antitumor compounds for a long term and screened out an indole alkaloid, 3a-acetonyltabersonine, had competitive antitumor effect in lung cacer, breast cancer and other cancer cell lines, but lacked data on glioma. Therefore, the thesis focuses on the research of the pharmacological effect and mechanism of 3a-acetonyltabersonine in glioma. 3a-acetonyltabersonine, which was isolated from Melodinus suaveolens, was found appeared remarkable cytotoxicity on glioblastoma stem cells and glioblastoma cell lines at low concentration. Furthermore, 3a-acetonyltabersonine could inhibit proliferation of glioblastoma stem cells and induce its apoptosis, which is nearly no similar report in other indole compounds so far. For this reason, 3a-acetonyltabersoninehas impressive advantage and research value. We found the effect of 3a-acetonyltabersoninerelated to phosphorylation and activation of MAPK signaling pathway through bioinformatics analysis of transcriptome sequencing and we confirmed these results by Western blotting. Under 3a-acetonyltabersonine treated condition, the expression of γ-H2AX and ATM was upregulated and the comet assay displayed typical feature,which revealed the role of 3a-acetonyltabersonine may correlate to DNA damage. Further research found that 3a-acetonyltabersoninedidn’t directly cause DNA damage but inhibited DNA damage repair process. Namely, the mechanism of 3a-acetonyltabersoninecan be summarized as follows: 3a-acetonyltabersonine inhibits DNA damage repair ability and results in DNA damage accumulation in a short time, then activates the MAPK signaling pathway and eventually leads to apoptosis.In order to further investigate the antitumor effect of 3a-acetonyltabersonine on glioma in vivo, we conducted experiments on mouse model. The tumor mouse model of glioma induced by lentivirus was confirmed by pathological analysisand the characteristics of glioma were significant. The experiment was carried out in a mouse model of glioma by in situ tube implantation. The mice, which were undergone drug administration had longer survival period. Furthormore, the immunohistochemistry results also showed that the apoptotic cells increased significantly in mice brain slicesafter3a-acetonyltabersonine treated.