【作者】 刘彦威；

【导师】 赵德明； 苏敬良；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2005， 博士

【摘要】 天然冬虫夏草是一种名贵的中药,资源短缺。为此,本实验探讨了人工冬虫夏草无性型的培养;同时,对无性型进行了鉴定,测定了其有效成分,并探讨了人工冬虫夏草抗肿瘤机制,为人工冬虫夏草替代天然冬虫夏草奠定了基础。 收集冬虫夏草子实体弹射的子囊孢子,将单个萌发孢子接种到培养基上,成功分离到冬虫夏草菌。同时观察了温度、pH值、通气、碳源、氮源和摇菌对菌丝生长特性的影响,结果显示冬虫夏草茵丝体为需氧菌,生长缓慢,可自发荧光,能分泌棕褐色样物质,最适温度为15～18℃,pH值范围为5～7,适当的摇菌可提高菌丝量,碳源和氮源对菌丝量有影响,适宜的碳源为淀粉、葡萄糖和甘露醇,最佳的氮源为复合氮源;用菌丝体和子囊孢子饲喂蝙蝠蛾幼虫可获得较高的僵虫诱导率(70～76.6%);免疫组化染色显示菌丝体和僵虫体内的菌丝有共同的抗原。这些为冬虫夏草人工培养、营养的选择及用免疫方法分析提供了有价值的资料。 采用PCR及DNA测序的方法,对不同产地的冬虫夏草及其分离株和其相关种类的rDNA ITS和5.8S RNA全序列进行分析,结果表明冬虫夏草和其分离株的序列相似性为97.5～100%,分离株与中国被毛饱相似性为98.4～99.7%而与Stachybotry chartarum、Cordyceps militari、Tolypocladium caledonica、Cephalosporium maydiss、Paecilomyces sinensis、Verticillium bulbillosum、相似性最高分别为78.9%、76.7%、72.2%、68.6%、62.8%、59.5%,这些结果提示冬虫夏草和分离株是同一品种的不同生长阶段,冬虫夏草为有性阶段,而其分离株为其无性阶段,即为中国被毛饱。 用比色法测定了天然冬虫夏草、人工培养的菌丝体和北虫草甘露醇的含量,探讨了摇菌和不同氮源对冬虫夏草菌丝体甘露醇含量的影响。结果显示天然冬虫夏草的甘露醇含量高于菌丝体,而菌丝体的含量又略高于北虫草;摇菌培养使菌丝和菌液中甘露醇含量降低:不同氮源培养基对菌液液和菌丝中的甘露醇有不同的影响,最佳氮源为复合氮源。提示天然和人工冬虫夏草均含有有效成分—甘露醇,人工冬虫夏草甘露醇的含量受培养方式和营养的影响,同时,这也为人工冬虫夏草替代天然冬虫夏草提供基础数据。 硫酸—苯酚法测定了天然冬虫夏草和菌丝体多糖含量及振荡培养和不同氮源培养基对菌丝甘露醇的影响,结果发现菌丝体的多糖含稍高于天然冬虫夏草(p>0.05);振荡培养抑制菌丝多糖产生,使菌丝多糖含量降低;氮源对菌丝多糖含量有影响,较适合的氮源为复合氮源。提示冬虫夏草多糖的含量受培养方式和营养的影响,同时,也为人工冬虫夏草替代天然冬虫夏草提供基础数据。 为了探讨冬虫夏草菌丝体水提液和多糖抗肿瘤细胞(SP2/0)作用机制,应用MTT法检测细胞增殖,琼脂电泳和流式细胞仪及Hoechst33342荧光染色观察和分析了细胞凋亡,流式细胞仪分析了细胞周期,实时荧光定量RT-PCR方法检测了不同处理样品p53基因表达变化。结果发现用冬虫夏草菌丝体水提液和多糖处理培养SP2/0细胞能抑制其生长,细胞分裂被阻滞在G1/G0期,且诱导细胞凋亡,p53基因表达下调,提示由于p53基因表达的变化,引起细胞周期阻滞,导致细胞凋亡,从而抑制细胞的增值。更多还原

【Abstract】 In this study, the culture of Annmorph of Cordyceps sinensis was discussed because of its expense and rare resources, while Annmorph was identified, efficient component was tested, and anti-tumor mechanism of Cordyceps sinensis was analysed, thus making solid basis for substitution of crude Cordyceps sinensis to artificial medcine.Several strains of annmorph of Cordyceps sinensis were successfully isolated through the seeding of budding ascopes derived from collection of several sporophores of Cordyceps sinensis. The culture condition of mycelium was tested. The results showed that mycelium of Cordyceps sinensis are aerobic, have very slow growth rate, brown secretion and auto-fluorescence. The optimal temperature for growth is 15~18℃, with pH 5~7. These provide valued refences for culture of Cordyceps sinensis, culture medium selection and immuno-analysis.There is mycelium weight increase in 0~16 days by flasks culture. The nitrogen and carbon sources have effect on mycelium weight. The better carbon sources are glucose, starch and mannitol. Among all nitrogen sources nitrogen-compounds source was the best. Larva of the Hepialu armoricamus was successfully infected by feed soaked with hyphae and ascopes; muscardine cadaver rate was 70-76.6 percent. There is same antigen of hyphae in the anamorph and the larva of the field infected Hepialu armoricamus by immuhistochemical method.These indicated that crude and artifical Cordyceps sinensis has some contents of efficient component, mannitol, that contents are affected by culture method and nutrition, so providing valued data for substitution of crude Cordyceps sinensis to artificial medcine.In this study, the rDNA ITS and 5.8SRNA of Cordyceps sinensis and its related taxa were amplified and sequenced The sequence comparison showed that similarity is 97.5%~100% between Cordyceps sinesis and A,B,C isolates, whereas similarity between Cordyceps sinesis and Stachybotry chartarum, Cordyceps militari, Tolypocladium caledonica,, Cephalosporium maydiss, Paecilomyces sinensis,Verticillium bulbillosum,were 78.9%, 76.7%, 72.2%, 68.6%,62.8%, 59.5%, respectively. Similarity between these isolates and Hirsutella sinensis were 98.4%~99.7%. Taken together, the three isolates and Cordyceps sinesis were same organism within different life cycle stages. These isolates were therefore the anamorph of Cordyceps sinesis, namely Hirsutella sinensis, rather than other species.The mannitol content of crude Cordyceps sinensis, cultured mycelium of cordyceps sinensis and Cordyceps militaris was measured via colorimetric method. While the effects of flasks culture and nitrogen soureces on mannitol content were also analyzed. The result showed that the mannitol content in crude Cordyceps sinensis was higher than that in culture mycelium, while which slightly higher than Cordyceps militaris culture. Mannitol content in mycelium and culture liquid decreased with flasks culture. The different nitrogen sources have various effects on mannitol level in mycelium and culture liquid, Nitrogen-compounds source was the best nitrogen sources. These indicated that mannitol contents are affected by culture method and nutrition, so providing valued data for substitution of crudeCordyceps sinensis to artificial medcine.The polysaccharides in crude cordyceps sinensis and mycelium were measured using phenol-sulphuric acid method, the effect of flasks culture and nitrogen sources on mannitol content were tested. The results indicated that the content of polysaccharides in mycelium was higher than that in crude Cordyceps sinensis. The content of polysaccharides was inhibited as result of decrease of mycelium level by flasks culture. The content examination of regulatory aspects on polysaccharides by different nitrogen showed that nitrogen-compounds was appropriate source. These indicated that polysaccharide contents are affected by culture method and nutrition, so providing valued data for substitution of crude Cordyceps sinensis to artificial medcine.To investigate the anti-tumor mechanism on SP2/0 myeloma cells by water extracts and polysaccharides from mycelia of Cordyceps sinensis, the inhibitory ratio of the cells were measured by MTT assay; apoptosis by agarose gel electrophoresis, flow cytometry (FCM) and hoechst 33342 fluorescence staining; cell cycle by FCM, and the expression of p53 gene was analyzed by real time RT-PCR in both the test and control groups. The results indicated that water extract and polysaccharides suppress the growth of SP2/0 myeloma cell line, induce cell apoptosis and phase G0/G1 arrest, and the level of p53 was decreased. These suggest that the change of p53 gene expression induced apoptosis and phase arrest of SP2/0 cells. The proliferation of SP2/0 myeloma cell inhibited through apoptosis and phase arrest.更多还原