【作者】 王薇；

【导师】 茆灿泉；

【作者基本信息】 西南交通大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 药用真菌用于治疗人类疾病有着悠久的历史,由于其具有高蛋白、低脂肪、低热量以及独特的优越性,近年来更被国内外学者关注。虫草、灵芝与猴头三种珍稀的食药用真菌,据研究显示具有抗肿瘤、抗氧化、提高免疫力、调节新陈代谢等多种药用。但野生虫草与猴头资源稀缺,为更好地开发药用真菌资源,使用液体发酵菌丝体己成为替代资源的一条重要的途径。但由于虫草的复杂生活史,以及系统发育地位的混乱,如何正确地鉴定虫草无性型已成为虫草液体发酵并开发其药用功效的首要条件。本研究采用PCR及DNA测序的方法,对13株不同产地的虫草分离菌ITS区序列进行分析,确认ITS作为条形码的可行性,成功鉴定了供试分离菌。同时采用生物信息学方法对Genbank中收集的虫草属36个物种以及肉座菌目的191个物种的ITS区序列进行多序列比对,构建进化树等分析,探讨虫草的系统发育地位及与相关物种的近缘关系。随后,本研究通过对已鉴定的虫草无性型,猴头,灵芝三种药用真菌进行液体发酵培养,并通过破碎,胶体磨粉碎,超声破壁等方法制备复合营养液,并通过DNS法测定了营养液中的多糖含量。,为了探讨复合营养液的药理作用,本研究采用MTT细胞活力测定,DNA梯状电泳,荧光染色等方法分析了复合营养液对MG63,Hela,HepG-2等体外肿瘤细胞凋亡的诱导作用。另外研究了复合营养液对过氧化氢造成的PC12细胞氧化应激损伤的保护作用,并测定其NO水平和LDH活性的变化。主要研究结果：1.通过ITS条形码鉴定分析,确认了CM-m5、CM-m7、CM-ml、 CM-m3和CM-m8为蛹虫草的无性型；CS-2和CS-kl为蝙蝠蛾拟青霉。2.成功制备了虫草复合营养液,采用DNS方法测定还原糖与总糖的方法测定了营养液中含有的多糖含量为22.52mg/ml,并考察了DNS法的稳定性,重复性及加样回收率。3.复合营养液能够有效抑制MG63、Hela等肿瘤细胞的增殖,并呈剂量依赖性,同时在高剂量营养液的作用下,可抑制HUVEC细胞的增殖,而在低剂量作用时,营养液可促进其生长,但对HepG-2肝癌细胞的抑制作用在48小时内并未体现。通过荧光双染以及DNA Ladder等指标检测凋亡作用,显示复合营养液可通过诱导MG63细胞凋亡从而达到抗肿瘤的效果。4.使用过氧化氢对PC12细胞进行造模处理,最终确定最佳的H202浓度为400μM,作用时间为24小时。通过MTT实验确认了当加药浓度在5%以下时为复合营养液对PC12细胞的安全浓度,在此基础上考察复合营养液对神经细胞氧化应激的保护作用,结果表明复合营养液能够有效地降低细胞上清LDH活性及NO含量,有效地减轻氧化损伤。主要结论：1. ITS序列可以作为虫草条形码对虫草及其相关物种进行鉴定以及系统发育分析,并从分子水平上验证虫草与其无性型之间的对应关系,确认了虫草属被划分为肉座菌目一员的说法,但与虫草与麦角菌科的从属关系相矛盾。2.复合营养液含有较为丰富的蕈菌多糖,在一定剂量内能够有效抑制肿瘤细胞及与肿瘤生长有关的血管内皮细胞的增殖,并诱导其凋亡,为后续的体内抗肿瘤工作打下基础。3.复合营养液可通过降低自由基NO水平以及减少LDH渗漏等方式对氧化应激损伤的PC12细胞产生保护作用,也为后续的抗神经退行性疾病作用提供了有效的细胞实验依据。更多还原

【Abstract】 Medical fungi in the treatment of human disease has a long history. In recent years, more and more scholars pay close attention to them, because of their high protein, low fat, low calorie, and unique advantages. Research shows that they have a variety of pharmacological effects on anti-tumor, anti-oxidation, enhance immunity, and regulation of metabolism. However, wild Cordyceps sinensis and Hericium erinaceus are scarcity resources. In order the better development of the medicinal fungi resources, mycelium of liquid fermentation has become an important way to get alternative resources. However, due to the complex life history and the confused phylogenetic status of the Cordyceps, the correct identification of Cordyceps anamorph has become the first condition for the Cordyceps liquid fermentation and the development of its pharmacological properties.In this study, PCR and DNA sequencing methods were used to analyze the isolated ITS sequences, confirm the feasibility of ITS as a barcode and identified13isolated strain from different origin. Multiple sequences alignment of ITS region sequences for36species of Cordyceps genus and191species of hypocreales from Genbank, phylogenetic tree analysis were used to investigate the phylogenetic position of Cordyceps and related species by bioinformatics. In this study, Cordyceps militaris, Hericium erinaceus, and Ganoderma lucidum were carried out liquid fermentation culture. The complex nutrient liquid was prepared by the method of the broken, colloid mill grinding, ultrasonic broken. Polysaccharide content of the nutrient liquid was determined by the DNS method.In order to investigate the pharmacological effects of the complex nutrient liquid, the MTT cell viability determination, DNA ladder electrophoresis, staining were used to analysis the induction of apoptosis to the MG63, Hela, HepG-2cells in vitro. In addition, protective effect of the complex nutrient liquid in PC12cells caused by oxidative stress injury of hydrogen peroxide was researched, The changes its level of NO and LDH activity were determined.Major results:1. Through the ITS barcode identification, CM-m5, CM-m7CM-ml, CM-m3, CM-m8were confirmed for the anamorph of Cordyceps militaris and CS-2and CS-kl were Hepialidae Paecilomyces.2. The complex nutrient liquid was successfully prepared. Polysaccharide content for22.52mg/ml and the stability, reproducibility and sample recovery of the DNS method were studied.3. The complex nutrient liquid can effectively inhibit the proliferation of MG63and Hela celsl, showing a dose-dependent. High levels of complex nutrient liquid could inhibit the proliferation of HUVEC, but the low-dose one can promote the growth of HUVEC. The complex nutrient liquid does not reflect the inhibition of HepG-2hepatoma cells within48hours. Fluorescent double staining and DNA ladder showed that complex nutrient liquid induced apoptosis in MG63cells to achieve anti-tumor effect.4. Hydrogen peroxide was used in PC12cells modeling, the optimal concentration of H2O2was400μM, for24h. The safe concentration of the composite nutrient solution in PC12cells was dosing concentration below5%, confirmed by MTT assay. On this basis, the protective effect of The complex nutrient liquid on the nerve cells to oxidative stress was examined. The results showed that the complex nutrient liquid can effectively reduce the cell supernatant LDH activity and NO content, and reduce the oxidative damage.Main conclusions:1. ITS sequence could be used as a barcode to identification and phylogenetic analysis in Cordyceps and its related species. Verify the correspondence between Cordyceps and its anamorphs in the molecular level and confirm that Cordyceps is divided into Hypocreales.2. The complex nutrient liquid has rich mushroom polysaccharide, which inhibit the proliferation of tumor cells and endothelial cells in tumor growth and induce apoptosis with a certain dose. That lay the foundation for the subsequent study in vivo.3. The complex nutrient liquid can produce a protective effect on PC12cells to oxidative stress injury by reducing the free radical NO level and the LDH leakage. Also provide an effective experimental basis for the next study, which can be used in anti-neurodegenerative diseases.更多还原