【作者】 刘春卉；

【导师】 瞿伟菁；

【作者基本信息】 华东师范大学 ， 植物学， 2007， 博士

【摘要】 金耳（Tremella aurantialba）为担子菌纲（Basidiomycetes），银耳目（Tremellales），银耳科（Tremellaceae），银耳属（Tremella）的寄生真菌，主产于我国西南部，野生资源稀有。为充分利用金耳资源，拓展创新药物研究与开发，对金耳生药学和发酵产物的活性进行系统研究，采用生药分子鉴别和病理模型等现代生物技术，探寻金耳单倍体无性酵母和寄主菌丝体鉴定及其产物生物学活性的证据。通过本草考证，认为金耳能够滋阴润肺，止咳化痰，生津。用于肺结核、虚痨、咳嗽、痰多、气喘、盗汗、高血压、糖尿病等症的治疗。经原菌物鉴定、显微鉴定、超微结构和培养特征研究，得到金耳生药学的基本特征：完整担子果呈脑状，剖面外层为橙黄色胶质，裂片中间为白色的肉质区，即寄主菌丝区，寄主菌丝含量比其它近似种高。菌丝细胞间有明显的隔膜，隔膜上有孔帽状结构，该结构可用以区别金耳菌丝和寄主革菌菌丝；寄主菌丝多核，有明显的锁状联合；子实层外露，成熟下担子为十字形纵分隔，顶生，上担子顶部膨大；担孢子球形，有小尖；担孢子反复出芽繁殖产生大量外被荚膜的分生孢子；担子果中有明显的吸器菌丝。通过对多个担子果的观察，发现以上特征很稳定，可以作为菌物分类和生药鉴定的重要依据。研究了金耳基源担子果、酵母状分生孢子培养物和菌丝培养物的核糖体DNA内部转录间隔区（ITS）和大亚基D1／D2区序列，结果表明金耳担子果的ITS区和D1／D2区的PCR产物均为碱基数不同的两条带，片段长度和序列与酵母状分生孢子培养物、菌丝培养物一致。通过对ITS1和ITS2联合进行系统发育分析表明金耳酵母状分生孢子培养物属于银耳属的金耳T. aurantialba，参与组成担子果的寄主菌丝为毛韧革菌Stereum hirsutum。大亚基D1／D2区系统发育分析结果与ITS区一致。结合GenBank中登录的金黄银耳、脑状银耳、橙黄银耳等近似种构建系统发育树，结果支持形态学证据，金耳药材和近似种金黄银耳形成一个稳定的独立分支，近似种黄金银耳形成另一个分支。分子序列的差异为金耳的鉴别提供了可靠的分子标记，为金耳菌类药材基源入药建立了遗传基础。首次报道了金耳的rDNAITS完整序列，包括ITS1和ITS2总长度为467-468 bp；5.8S序列为158 bp以及大亚基D1／D2区序列（638 bp）。金耳寄主粗毛韧革菌的rDNA ITS完整序列，包括ITS1和ITS2总长度为591-593 bp；5.8S序列为158 bp以及大亚基D1／D2区序列（607 bp）。金耳与寄主粗毛韧革菌的5.8S区序列同源性为97％。对人工栽培金耳药材ITS及5.8S区和D1／D2区序列研究结果表明，栽培金耳存在种内变异性。测定了收集的金耳试管菌株ITS区和D1／D2区序列，BLAST比对，发现存在误将寄主菌丝株作为金耳菌丝的现象，认为金耳对于伴生菌的依赖性在于担孢子形成的单一型金耳菌丝对基质中的养分分解能力极弱，必须借助毛韧革菌丝来获得营养物质，因而导致人工培养金耳时不仅难以获得纯培养的金耳菌丝，而且由于金耳菌丝的生长劣势易被伴生菌丝取代，同时也表明金耳制种时采用分子标记法进行鉴别较为可靠。采用金耳无性酵母菌株B1，对其产生胞外多糖（Extracellular Polysaccharides，EPS）的液体培养基配方进行研究，以获得最优的液体培养基。培养基优化结果表明葡萄糖是菌体生长和EPS产生的最佳碳源，中心组合设计和响应面分析得到最佳碳源为49.2 g／L的葡萄糖，最佳氮源为10.4 g／L的酵母膏的优化配方，最大EPS产量为5.02 g／L。优化前胞外多糖产量为3.43 g／L，优化后的EPS产量是优化前的1.5倍，明显提高了菌株B1的生物学效率。金耳EPS对STZ诱导的胰岛素抵抗实验动物模型空腹血糖有显著降低作用，0.2 g／kg·BW／d剂量，连续灌胃EPS 21 d后空腹血糖降低30.1％，口服糖耐量2h血糖显著降低，果糖胺水平下降59.5％，胰岛素敏感指数显著升高，胰岛素抵抗指数显著降低，胰岛素水平未见显著下降，提示其可能通过调节胰岛素以外的其它途径改善胰岛素抵抗。给EPS组的胰岛素抵抗大鼠的TG水平和LDL-C水平显著降低，HDL-C水平显著升高，相关的LPL和HL的活性也显著升高，表明由胰岛素抵抗引起的脂代谢异常也得到改善。通过对金耳无性酵母液体培养耐受和富集三价铬(Cr³⁺)能力的研究，依据菌种的生理特性建立了富铬金耳无性酵母菌体的基础发酵条件：碳源采用葡萄糖，氮源采用酵母膏，基质Cr³⁺添加浓度50μg／mL，发酵周期以66-72h为佳，获得了Cr³⁺含量为2.87 mg／g干重的富铬金耳菌体，Cr³⁺含量是文献报道的富铬啤酒酵母（1.5mg／g干重）的1.91倍；是富铬子囊菌Cr³⁺含量（0.8 mg／g干重）的3.59倍。大鼠急性毒性实验结果表明口服富铬金耳无性酵母菌体在最大给药量为11g／kg·BW／d未见急性毒性反应。30d喂养试验结果表明，口服富铬金耳菌体各剂量组大鼠均未见异常症状和体征，也无死亡；对大鼠的体重、进食量、食物利用率及生长发育未见明显影响；红细胞计数、血红蛋白、白细胞计数等血液学指标也未见明显影响；富铬金耳各实验组与对照大鼠的血清葡萄糖、白蛋白、胆固醇、尿素氮、谷草转氨酶、谷丙转氨酶、总蛋白、甘油三酯、肌酐等血液生化学指标检查结果均在正常范围，脏器组织病理学检查结果未发现异常改变。富铬金耳对正常大鼠的无作用剂量在7.92 g／kg·BW／d以上，为人体推荐摄入量的100倍以上。STZ诱导的胰岛素抵抗实验动物分别按1.32 g／kg·bw／d、0.66 g／kg·bw／d、0.33 g／kg·bw／d灌胃富铬金耳21d后，三个剂量组均有降低模型大鼠空腹血糖和口服糖耐量血糖的作用，果糖胺水平也显著降低，与盐酸二甲双胍降糖作用比较无显著性差异，但胰岛素水平未见显著降低，中剂量和高剂量组的胰岛素敏感指数显著升高，胰岛素抵抗指数显著降低，初步认为富铬金耳能够改善实验性胰岛素抵抗大鼠模型的口服糖耐量，能够显著降低血糖和果糖胺水平，虽未改变胰岛素的分泌功能，但显著提高了胰岛素敏感指数，从而显著改善胰岛素抵抗指数。采用金耳寄主粗毛韧革菌发酵法制备以萜类为主的小分子混合物SHE（Ethanol extract from the fermented mycelium of Stereum hirsutum，host fungus of T. aurantiabla），观察SHE对体外培养的人肾透明细胞癌（786-O）和人肝癌细胞（BEL-7404）的细胞毒作用。MTT法结果表明SHE能够抑制786-O和BEL-7404细胞的生长，抑制作用随SHE浓度的升高而增强。SRB法蛋白含量测定结果发现SHE作用48 h后，786-O细胞与SRB结合的蛋白质含量显著下降，IC₅₀为523.13μg／mL。Wright染色和结果显示，786-O经SHE作用后呈现典型的凋亡形态学改变。流式细胞技术研究发现SHE组出现明显的凋亡峰。SHE作用48h后，BEL-7404细胞与SRB结合的蛋白质含量显著下降，IC₅₀为567.38μg／mL。Wright染色结果显示，BEL-7404经SHE作用后呈现典型的凋亡形态学改变。流式细胞技术研究发现SHE组也出现明显的凋亡峰。更多还原

【Abstract】 Tremella aurantialba, one species of Basidiomycetes, Tremellales, Tremellaceae, Tremella, was a kind of mycoparasites and distributed mainly in southwest of China. And it was protected as a wild scare resource. With the aim to utilize ultimately the resource of T. aurantialba and further to develop innovative medicine research, pharmacognosy of crude herb and bioactivities of the fermented products of T.aurantialba were investigated systematically, meanwhile, the present study adopted some modern biotechnologies, such as the molecular identity of crude fungus and pathologic experimental animal models, and the results would be beneficial for developing new drugs from T. aurantialba fungus.According to the traditional Chinese medicinal literatures , basidiocarps of T.aurantialba were used as a kind of salubrious and beneficial herb for nourishing yin, moistening lung and cough treating, therefore, it was used for treatment of tuberculosis, cough, hypertension, and diabetes etc. Based on morphological survey, microscopic and ultrastructural identification and cultural characteristics, the basic medicine features of this species were discovered. Basidiocarps cerebriform when mature, almost foliaceous, the lobes rugose and plicate, giving the surface a strongly wrinkled appearance; in section, lobes with a white fleshy interior zone of host hyphae, the outer portions gelatinous, orange, amounts of its host hyphae high than that of other related species. The hyphae of T. aurantialba have appearant septa with the pore caps. The host hyphae mixing in the basidiocarps of parasites can be easily distinguished by their septal pores, coenocytic cells and abundant clamps. Bare hymenium exist on the surface of basidiocarps; mature basidia cruciate-septate, acrogenous; cells immediately below basidia often swollen; basidiospores globose with apiculus, germination by repetition or by budding, hymenial condia produced many yeast cells with outer capsule; abundant haustoria hyphea in the inner part of basidiocarps. Besides the above features were relatively stable, therefore, these features of the species can be the taxonomic and pharmacognosy characteristics for certification.The sequences of rDNA ITS and 28S D1/D2 region of the basidiocarps, anamorph blastospores and mycelia isolates from T. aurantialba were studied. The results indicated that the PCR products of ITS and D1/D2 region sequences contained two bands with different number of bases: one was identical with sequences of the anamorph blastospore isolates, and the other was identical with sequences of the mycelium isolates. Phylogenetic analysis based on ITS1 and ITS2 sequences indicated that the anamorph blastospore isolates is T. aurantialba, while the mycelia isolates, the host fungus of T. aurantialba, is Stereum hirsutum. The results of phylogenetic analysis based on D1/D2 regions sequences were consistent with the results of ITS sequences. Phylogenetic trees were constructed by using the above sequences of T. aurantialba and those of its related species which were T. aurantia, T. encephala and T. mesenterica registered in GenBank. T. aurantialba and T. aurantia formed a separate stable branch in the topology trees, while T. mesenterica clustered on another clade, which is consistent with the morphological evidence. The characteristics of ITS and D1/D2 region sequences can be used for molecular identification to distinguish the analog species of the fungi, and providing the basic molecular biological information for further pharmacological study of fungal resources. It is the first report of the whole sequences of rDNAITS regions （including ITS1, 5.8S and ITS2） of T. aurantialba. ITS sequences was 467bp and 468bp, respectively; 5.8S sequence was 158bp,and D1/D2 region sequences was 638bp. The whole sequences of rDNA ITS regions of Stereum hirsutum were detected that ITS sequences were 591bp and 593bp, respectively; 5.8S sequence was 158bp,and D1/D2 region sequences was 607bp. 5.8S sequences of T. aurantialba had highly homologous with its host Stereum hirsutum.The sequences of rDNA ITS and 28S D1/D2 region of the artificial cultivated basidiocarps were studied. The comparison results of these sequences showed that there exsisted intraspecies variations in the artificial cultivated and wild T. aurantialba. The sequences of rDNA ITS and 28S D1/D2 region of T. aurantialba mycelium strains collected from different institutes were determined and BLAST. The results were clearly divergent from T. aurantialba, but closed related to S. hirsutum. We presume that host hyphae in pure cultures were misunderstanded as hyphae of T. aurantialba. Consequently, Use of the database and the method of molecular marker could have particular value for the rapid identification of species in cultures.Both the morphological certification and the phylogenetic analysis had confirmed that fungus B1 was one of the anamorph strains of T. aurantialba. The optimization of submerged culture conditions for cell growth and exopolysaccharides （EPS） production in T. aurantialba fungus B1 were studied in shaker-flask system. First, a Plackett-Burmen design was used to evaluate the effects of different components in the culture medium. Glucose and yeast extract have significant influences on the EPS production. The concentrations of two factors including in glucose and yeast extract were optimized subsequently using central composite designs and response surface analysis. The optimized medium led to a 1.5-fold enhancement of the production of EPS （5.02 g/L） by T. aurantialba fungus B1, when compared to the unoptimized medium in which the yield of EPS is only 3.43 g/L.The hypoglycemic effect of EPS produced from submerged culture of T. aurantialba fungus B1 in streptozotocin-induced insulin resistant type 2 diabetic rats was investigated. The fasting blood glucose levels in diabetic rats were substantially reduced by 30.1% in the diabetic rats by continuous oral-douche of EPS with a dose of 0.2 g/kg-BW/d for 21 days compared with the diabetic rats served as controls. Moreover, the 2-h postprandial blood glucose concentrations in an oral glucose tolerance test （OGTT） were significantly attenuated by EPS gavage in the diabetic rats, and the serum concentration of fructosamine were decreased by 59.5% . Although EPS had no influence on serum insulin levels, insulin sensitivity index and insulin resistance index showed the statistical difference in oral-EPS diabetic rats. The above results suggested that EPS had a potential hypoglycemic activity. Furthermore, EPS lowered the serum triglyceride and LDL-C concentrations, while increasing the levels of HDL-C, the activities of lipoprotein lipase, and hepatic lipase in diabetic rats. Therefore, it could be concluded that EPS can ameliorate the lipid metabolism in insulin resistant diabetic rats.The use of the anamorph yeast of T. aurantialba as the vector for chromium（III） bioaccumulation, Cr-tolerance, and Cr-accumulating procedures of the yeast biomass in liquid fermentation were studied. The basal fermented culture medium was determined systemically and obtained. The glucose and yeast extract were used as the carbon and nitrogen source, respectively. When Cr³⁺ concentration were added to 50μg/mL in liquid culture medium, the maximum chromium（III） content 2.87mg/g dry biomass was obtained at the 66-72 h. The Cr³⁺content was 1.91-fold and 3.59-fold enhancement by T. aurantialba yeast, as compared by chromium-enriched brewer’s yeast and ascomycete of the literatures, respectively.The acute and 30d chronic feeding toxicological experiments were carried out to evaluate the toxic effect of chromium-enriched yeast of T. aurantialba. Acute oral toxicity test to adult male and females rats indicated that there was no acute toxic effect even if the chromium-enriched yeast were given by gavage to the maximum dose of 11 g/kg·BW/d. And the 30 day feeding test in normal rats showed that chromium-enriched yeast of Tremella aurantialba had no significant influence on food intake, body weight, viscera /body weight ratio, number of red and white blood cells and the content of hemoglobin; the function of liver and kidney and the ability of lipid and protein metabolism were considered in normal ranges; and no dead rats was recorded in each group. The 30-day oral non-effect dose in normal rats was up to 7.92g/kg·BW/d which was equal to 100 times of the dose for human.A 21 days experiment was studied to investigate the anti-diabetic effect of chromium-enriched yeast of T.aurantialba in streptozotocin-induced insulin resistant type 2 diabetic rats models by oral douche （ 1.32 g/kg·bw/d, 0.66 g/kg·bw/d, 0.33 g/kg·bw/d, respectively）. The results showed that the fasting blood glucose concentrations in three dose groups of diabetic rats were lowered, respectively. The chromium-enriched yeast of T.aurantialba remarkablely attenuated the elevated 2-h postprandial blood glucose levels in an oral glucose tolerance test （OGTT） , and the serum fructosamine levels in the diabetic rats, While significant differences were not be observed in the hypoglycemic effect between the chromium-enriched yeast of T.aurantialba and the Metformin hydrochloride tablets. Though chromium-enriched yeast of T.aurantialba had no influence on serum insulin levels, the insulin sensitivity index and insulin resistance index showed the statistical difference between diabetic rats of chromium-enriched yeast of T.aurantialba dose groups （0.66 g/kg·d or 1.32 g/kg·d） and the diabetic rats models. The above results confirmed again that the chromium-enriched yeast of T.aurantialba had a potential hypoglycemic activity.The ethanol extract （SHE）, with the main components of which were the the terpenoid compounds, was isolated from the fermented mycelium of Stereum hirsutum （host fungus of T. aurantiabld）, and its anti-tumor activities were investigated. The cytotoxicity of SHE on two human tumor cell lines renal neoplasm cell 786-0 and hepatoma cell BEL-7402 was observed in vitro, respectively. The results of MTT assay showed that SHE significantly inhibited the growth of 786-0 and BEL-7404 cells, and the inhibitory effects increased with the SHE concentration treated increasing. And the results of SRB protein content exhibited significiant cytotoxity effects （P<0.05） on 786-0 and BEL-7404 cells, at 48 h incubation. The IC₅₀ of SHE on the cytotoxicity of 786-0 was 523.13μg/mL. The results of morphological changes by using Wright acridine orange stain and propidine iodide stain assay showed that SHE induced the typical morphological characteristics of apoptosis on 786-0. 786-0 Cells treated with different concentration of SHE exhibited remarkable apoptosis apex by observation of the flow cytometry detection. The results of SRB protein content assay, the morphological observation by Wright stain and propidine iodide stain assay, and the flow cytometry detection on BEL-7404 cells were similar with those on 786-0, while the IC₅₀ of SHE on the cytotoxicity of BEL-7404 was 567.38μg/mL.更多还原