【作者】 王建锋；

【导师】 苏文金；

【作者基本信息】 厦门大学 ， 动物学， 2001， 博士

【摘要】 植物内生真菌是指生活在植物组织体内的一类并不引起明显病害症状的真菌，包括了那些在其生活史中的某一阶段营表生的腐生菌，对宿主暂时没有伤害的潜伏性病原菌(Latent pathogens)和菌根菌(mycorhizafungi)。它是一个庞大的特殊的真菌类群，几乎所有的植物组织中都发现有内生真菌的存在。研究表明，在某些药物植物(如红豆杉)中，存在一些能够产生与宿主相同代谢产物(如紫杉醇)的内生真菌；而且由于真菌与植物之间长期的相互作用和共同进化，其代谢产物十分丰富，包括杀虫、抗菌和抗肿瘤等活性物质。因此，植物内生真菌现己成为寻找抗肿瘤药物的重要来源。 我国药用植物资源十分丰富，对其内生真菌的研究将有利于我国微生物药物的开发和珍稀植物资源的保护。本文以福建省几种珍稀药用植物为对象，研究其内生真菌抗肿瘤活性，目的在于确定抗肿瘤活性菌株的生态分布及其抗肿瘤特性，从而为植物—真菌生态关系的研究以及植物内生真菌资源的利用提供依据。 从福建武夷山、三明沙县等地区的南方红豆杉、香榧和三尖杉的树皮中共分离得到172株内生真菌。采用细胞毒筛选模型(MTT法)对这些内生真菌发酵液的抗肿瘤活性进行了检测。结果表明，共有25株内生真菌(占总供测菌株的14.5％)的发酵液对KB或HL-60细胞生长有明显的抑制作用(ID50≤1：50)。红豆杉、香榧和三尖杉内生真菌抗肿瘤活性菌株各占供测菌株的19.6％、8.6％和3.3％。抗肿瘤活性菌株主要分布于拟青霉属(28％)和头孢霉属(24％)等8个属中，同属真菌的抗肿瘤活性也有很大的差异。内生真菌种属和生物活性的多样性为寻找抗肿瘤活性代谢物质奠定了基础。 对两株分离自红豆杉和香榧的抗肿瘤高活性菌株W-TF5和H-036的抗肿瘤活性物质作了进一步的研究。采用TLC、UV、HPLC、MS等理化方法和MTT以及细胞外和细胞内微管聚合等生物实验手段证实了W-TF5发酵液有机抽提物中存在抗肿瘤药物紫杉醇；采用柱层析和重结晶等方法从W-TF5发酵液中分离到另一种抗肿瘤活性物质的结晶，X-Ray单晶衍射、IR、MS以及NMR等数据表明该晶体是一种11元环松胞菌素——松胞菌素D(Cytochalasin D，CytD)，首次确定了松胞菌素D的晶体结构。 H-036发酵液对HL60、KB、Hela、SPC-A-1和MCF-7细胞都具有 王建锋博士论文 摘 要、11． 很强的细胞毒作用，将其发酵液用乙酸乙酯抽提浓缩后，用甲醇、丙酮等 溶剂反复重结晶，即可得到无色纯净的柱状晶体。采用XRay单晶衍射、 IR以及NMR等手段确定了该晶体的是一种大环内酯类物质—一布雷菲德 菌素 A（Brefe旧in A，BFA）。 本文工作证实了南方红豆杉、香皿和三尖杉中存在着丰富的植物内生 真菌资源，种属多样。抗肿瘤活性菌株W－TFS除了产生紫杉醇外，还可 产生 CytD，经鉴定为一种瘤座抱（Tubercularia sp、X 这是首次从红豆杉 类植物中分离到瘤座抱属真菌，是一株紫杉醇和松胞菌素新的产生菌。 Ho36可产生 BFA（产量 31 00mglL发酵液人 经鉴定为一种拟青霉 （Paecilomyces sp．L是一株布雷菲德菌素新的产生菌。这些结果表明药 用植物内生真菌是研究和开发抗肿瘤微生物药物的宝贵资源。 本文还对分离到的CytD和BFA对肿瘤细胞的作用作了初步的探讨。 采用MTT和贴壁集落等方法检测了CytD和BFA对HL60、KB等肿瘤细 胞的抑制作用，并采用流式细胞检测法和激光共聚焦扫描形态观察法研究 了CytD和8＊A诱导细胞凋亡的作用。结果表明，Cy旧对H＊0、＊B、 ＊＊a、＊C＊7和即C－入1细胞的忆5。分别为25o、25o、250、500、10o nglml；BFA的细胞毒作用要比 CytD强，对这些细胞的 IC幼分别为 15、 10、3刀、2石、2刀 nglml。CytD能够诱导 HL60和 KB细胞 GZIM期阻滞 和细胞凋亡：经oytD处理的K日细胞在6 h 内就出现起泡现象〔0dD b旧bbing），在 24 h后 GZIM 期阻滞至达最高峰（18％上升至 40％），48 h 后 BC卜2的表达明显下降，大量细胞凋亡（4．8％上升到 55％）。提示微丝 系统对细胞信号的传导具有重要作用，对微丝的破坏，可能会诱导细胞凋 亡。BFA能够诱导HL60及KB细胞的G0lGI期阻滞和细胞凋亡。BFA处 理 KB细胞 24 h后，细胞不能由 G。期进入 S期，导致 G。IG；期阻滞，此 时只有个别的细胞发生凋亡，但 48 h后细胞即大量凋亡，并伴随着 BC12 的磷酸化。更多还原

【Abstract】 Endophytic fungi are the microbes associated with living tissue of plants without causing any noticeable symptoms of disease. They constitute a large, special fungal group, and they are to be found in all or almost all plants. It’s reported that some endophytic fungi could produce the same metabolite as that produced by their hosts. For example, some endopytic fungi isolated from Taxus sp. could produce taxol, one of most important antitumor drugs. Moreover, the secondary metabolites of endophytic fungi are abundant, including insecticidal, antimicrobic and antitumor agents. Therefore, endophytic fungi have become a new promising source for screening antitumor drugs.China has very rich pharmaceutical plants resources. Studies on endophytic fungi will promote the development of microbial medicine and help the protection of the valuable and rare plants. We have studied the antitumor activities of the endophytic fungi of several pharmaceutical plants in Fujian province in order to find new antitumor drugs.Altogether there were 172 strains of endophytic fungi isolated from the bark of three plants from Wuyi Mountain and Sanming County in Fujian province: Taxus mairei, Torreya grandis and Cephalataxus fortunei. The antitumor activities of the fermentation broths of these endophytic fungi were tested by MTT method. The result shows that 25 fermentation broths (about 14.5% of the total strains isolated) show noticeable growth inhibition against KB and HL6O tumor cells. The strains with antitumor activity in three plants, T. makei, T. grandis, C. fortunei account for 19.6%, 8.6%, 3.3% respectively among the total strains isolated. The strains with antitumor activity can be grouped into 8 genera by conventional identification. Paecilomyces (28%) and Cephalosporium (24%) were the main genera among the strains identified with antitumor activity. Even in the same genus, the antitumor activities show obvious difference. The diversity of genera and antitumor activities provides us with the possibility of searching antitumor metabolites.We further studied the antitumor metabolites of two strains withhigh antitumor activity, W-TF5 and H-036, which respectively isolated from T mairei and T grandis. Using TLC, UV, HPLC, MS, MIT, Tubulin Assembly methods, we verified that W-TF5 could produce taxol. From organic extract of W-TF5 fermentation broth, we also isolated another antitumor compound by column chromatography and recrystal methods, which was identified as Cytochalsin D (CytD) by X-Ray single crystal diffraction, IR, MS and NMR methods. Moreover, the X-Ray crystal structure of CytD was determined for the first time in the world.The fermentation broth of H-036 has a strong cytotoxicity on HL6O. KB. Hela~ SPC-A-1 and MCF-7 tumor cells. After the broth was extracted with ethyl acetate and condensed, and recrystalized in methanol and acetone repeatedly, a kind of colorless, pure columner shaped crystal was obtained, which identified as Brefeldin A (BFA) by X-Ray single crystal diffraction, lR and NMR methods.Our studies show that endophytic fungi are abundant in T mairei, T grandis and C. fortunei with great diversity. Strain W-1F5, identified as a Tubercularia sp., can produce both Taxol and CytD. This is the first time that finds Tubercularia sp. isolated from Taxus sp., and strain W-TF5 has been found as a new taxol and CytD-producing fungus. Strain H-036, identified as a Paediomyces sp., can produce BFA with high production (~10Omg/L), which is a new BFA-producing fungus. These results indicate that studies on endophytic fungi of pharmaceutical plants are beneficial to develop new microbial medicine.We also primarily studied the effects of CytD and BFA on tumor cell. CytD shows cytotoxicity on all tested tumor cell lines (HL60~ KB~ Hela. MCF-7 and Spc-A-1) with 1C50 ranging from 100 ng/ml to 500 ng/ml. BFA has stronger cytotoxicity than CytD with IC50 ranging from 2.0 ng/ml to 15 ng/ml. CytD can induce G2IM inhibition and apoptosis of HL6O and KB cells. After treated with CytD, KB cells appeared cell b更多还原