具有抗肺癌作用的三株真菌次生代谢产物及作用机理的研究

Preliminary studies revealed that three saline soil derived fungi (Penicillium raistrickii, Aspergillus iizukae and Aspergillus sclertoiorum) showed strong activity against lung cancer cell line. The three high valuable fungi need to be developed and investigated. Various chromatography techniques were used to isolate and purify the secondary metabolites from the fungi. The structures of the isolated compounds were elucidated by spectroscopic data and physicochemical properties. The cytotoxicities against A549 were assessed by use of the MTT method in vitro. Furthermore, the activities of the isolated compounds against A549 cell line in vivo was carried out by injecting the A549 cell line subcutaneously into nude mice. Intracellular reactive oxygen species (ROS) and potentials of mitochondria were studied on the basis of the cyto-immunofluorescence staining technique. The variation statuses of proteinum and correlation factor were detected using of PCR, Western-blot, and etc. In order to reveal the anti-tumor mechanisms of the compounds, the high-throughput gene chip method was used to search for the new factors and special targated molecules produced in the process of the active compounds inducing apoptosis of A549 cell line. The program will lay a foundation for the research for anticancer drugs and provide promising resource fungi with antitumor.

前期研究发现，三株来自盐碱地的真菌(Penicillium raistrickii, Aspergillus iizukae，Aspergillus sclertoiorum)具有很强的抑制A549细胞生长并诱导其凋亡的作用，具有进一步研究和开发的价值。本课题拟以现代色谱技术系统分离、纯化三株真菌代谢产物，利用波谱技术和理化性质确定单体化合物的结构；用MTT法测定单体化合物对A549细胞生长的抑制率，体内用裸鼠皮下接种肺癌A549细胞，观察其对肿瘤抑制率；对抗肺癌活性优良的化合物，用细胞免疫荧光染色法研究细胞内ROS和线粒体膜电位；应用PCR、Western-blot等方法检测蛋白和相关因子变化情况，使用高通量的基因芯片技术，寻找参与活性化合物诱导A549细胞凋亡的新因子，寻找其特异性靶标分子，揭示抗癌作用机制，为抗肿瘤药物研发奠定基础，为微生物抗癌药物的发现提供资源菌。

盐碱地作为一个特殊的生态环境，孕育着许多独特的微生物，盐碱地中的真菌受环境的影响可能产生结构独特、活性优良的化合物，课题组在前期工作中从滨州临海重度盐碱地中分离得到了3株（Penicillium raistrickii、Aspergillus iizukae和Aspergillus sclertoiorum）具有较强的抗肿瘤活性真菌。本课题用正反相硅胶柱、薄层层析、凝胶LH20、高压液相色谱等手段从这三株真菌中分离得到96个化合物，利用核磁共振谱、质谱、圆二色光谱(CD)、单晶X衍射等波谱技术和理化性质确定了它们的结构，其中新化合物30个，5个具有较强抗肿瘤活性的化合物；具体情况如下：课题对P. raistrickii菌进行了摇床发酵和静置发酵，从两种发酵条件下的代谢产物中分离得到74个化合物，其中新化合物24个；从A. iizukae中分离得到11个化合物，其中2个新化合物；从A. sclertoiorum菌中得到15个化合物，新化合物4个。. 课题以多种肿瘤细胞为模型，用细胞免疫荧光染色法研究细胞内ROS；应用PCR、Western-blot等方法检测蛋白和相关因子变化情况，研究了peneciraistin C和peniciketal A的抗肿瘤机制，为抗肿瘤药物研发奠定基础。. P. raistrickii菌的代谢产物类型非常丰富，有螺环缩酮、吡喃、吡喃酮、异香豆素、酸酐、甾醇、喹啉酮、咔啉生物碱、二肽、氯代物、醌类化合物等。课题考察了pH和盐度对P. raistrickii菌的影响，结果发现不同条件下该菌株发酵液的颜色、指纹图谱有较大的差别，说明pH和盐度对该菌的代谢产物有较大影响，该研究为进一步开发P. raistrickii奠定了基础。. 上述研究以第一或通讯作者在Org. Lett.、Mar. Drugs、Cancer Sci.等杂志发表SCI收录论文4篇，中文核心期刊1篇；获授权专利1项。以上工作按计划完成了本项目的各项指标。该研究为为深入研究、开发与利用药用盐碱地真菌新资源，推动创新药物的研究与开发奠定了基础。