分级组合筛选和单菌多产物策略下烟草内生真菌中抗肿瘤活性代谢产物研究

烟草内生真菌是未被充分发掘的新型抗肿瘤活性先导化合物的重要来源。然而单一的化学生物学的方法在初步筛选中存在假阳性及菌株重复研究问题。为了对潜力菌株进行稳定可靠的定向筛选，深入发掘活性代谢产物多样性，前期利用MTT法-光镜观察法-流式细胞术分级组合筛选及OSMAC策略，发现一株烟草内生真菌Fusarium sambucinum TE-6L具有显著抗肿瘤活性，且在不同培养条件下可产生多样性的代谢产物。据此，本课题拟继续以下研究：（1）利用OSMAC策略深入发掘不同培养条件下其活性代谢产物结构多样性；（2）利用HPLC-DAD及LC-MS/MS指纹图谱对发酵产物中特征活性化合物进行快速的发现、分离及结构解析；（3）利用分子药理学方法探讨活性化合物作用机理，为抗肿瘤创新药物提供先导物。本项目选取烟草内生真菌这一特殊资源为研究对象，从新的筛选思路出发，为开发利用植物内生真菌资源提供科学依据。

Tobacco endophytic fungi are un-fully exploited vital sources of new antitumor lead compounds for drug discovery. However, the simple chemicobiological method applied in the preliminary screening faces a number of technical challenges. These include the false positive reactions observed in the test, the repetitive research on the microorganism, as well as the overlapped discovery of the known compounds. To achieve stable and reliable screening of potential strains and thoroughly excavate the chemical diversity of the active metabolites, in our early experiments, the gradated-combinatorial screening based on MTT method, light microscopy, and the flow cytometric assay were employed. The culture strategy of OSMAC was also used to activate their metabolic potentiality. The results showed that the endophytic fungus Fusarium sambucinum TE-6L exhibited potent antitumor activity and it could produce diverse metabolites under different culture conditions. Based on our previous studies, in this project, (1) the culture strategy of OSMAC will be applied to thoroughly excavate the diversity of the active metabolites under different culture conditions; (2) The characteristic bioactive constituents will be isolated and purified under the guidance of the HPLC-DAD and LC-MS/MS spectra, and their structures will be further elucidated by spectroscopic methods; (3) The antitumor mechanism was discussed, by using the molecular pharmacological methods, so as to providing latent leading compounds for antitumor drug research. The tobacco endophytic fungi were studied for the first time, and the new screening strategy can help to find new antitumor lead compounds, which will provide scientific basis for the development and effective use of the fungal endophytes.