【作者】 查学强；

【导师】 罗建平；

【作者基本信息】 合肥工业大学 ， 农产品加工及贮藏工程， 2006， 博士

【摘要】 石斛作为传统名贵中草药，其资源再生，化学成分和药理活性逐渐引起人们的关注，多糖是石斛属植物中主要的药用活性成分之一。本文以传统的名贵珍稀药用植物——霍山石斛为主要研究对象，在建立霍山石斛类原球茎多糖高效表达悬浮培养体系的基础上，利用生物大分子研究的最新技术和现代分离分析手段与药理实验方法，对霍山石斛类原球茎水溶性多糖进行了提取分离纯化，并对其主要活性多糖组分HPS-1B23的组成和一级结构进行了研究。主要研究结果如下： （1） 以霍山石斛试管苗茎段为外植体，首次诱导获得了类原球茎，并建立了类原球茎的培养体系。其中附加NAA(7.5μmol L^-1)和KIN(0.5μmol L^-1)组合的MS培养基最利于类原球茎的形成，诱导率达到79.9％。诱导的类原球茎经长期继代培养后，从生长、植株再生、活性多糖合成、药理作用、单糖组成及基因组DNA多态性等方面进行品质稳定性分析，表明诱导获得的类原球茎系在品质上是稳定的。 （2） 药理学研究证明了培养的类原球茎具有野生霍山石斛同样的合成活性多糖的能力。野生霍山石斛总多糖能显著促进小鼠脾细胞和巨噬细胞分别产生IFN-γ和TNF-α，多糖作用浓度分别在800μg mL^-1和200μg mL^-1。RT-PCR分析表明，总多糖是通过上调或下调IFN-γ和TNF-α的基因表达来控制各自的释放量的。经DEAE-Cellulose离子交换色谱层析，从类原球茎总多糖中分离出和野生霍山石斛相同的5个多糖组分，并表现出同样的促进小鼠脾细胞和巨噬细胞分别产生IFN-γ和TNF-α的能力，其中以水洗脱部分HPS-1含量最大。建立的多糖活性评价方法实现了对霍山石斛类原球茎培养体系的质量监控。 （3） 研究分析了霍山石斛类原球茎液体培养过程中碳、氮代谢特征，阐明霍山石斛类原球茎生长和多糖合成与细胞内源和外源碳、氮的关系。在整个培养过程中，没有观察到显著的细胞生长延滞期，培养30d后，生物量达到最大，多糖合成与类原球茎生长表现出了非同步的关系。细胞培养启动后，在前3d培养基中蔗糖浓度减少了一半，同时无葡萄糖和果糖的存在，之后随着蔗糖浓度的继续下降，培养基中逐渐检测到有葡萄糖和果糖的存在，到第9d含量达到最大值。相反，类原球茎内源蔗糖随培养的启动，在培养的前6d，快速积累，之后快速被消耗；内源葡萄糖和果糖含量也同时有一定程度的增加，培养9d后逐渐被消耗。可溶酸性蔗糖酶和碱性蔗糖酶随类原球茎培养的启动逐渐被激活，分别在培养的第6d和第18d活性达到最大值。细胞壁蔗糖酶在整个培养过程中活性很低，一直处于被抑制更多还原

【Abstract】 Dendrobium species are used as the traditional Chinese medicine herbs for centries. Much attention has been focused on its resource regenesis, chemical components and pharmacological activities. Polysaccharides were the major active constituent from Dendrobium plants. Dendrobium huoshanense C. Z. Tang et S. J. Cheng, from which the Chinese medicine was most originally prepared, was studied in this paper. After the high expression system of polysaccharides from protocorm-like bodies （PLBs） in suspension culture was researched, water-soluble polysaccharides were extracted, isolated and purified from PLBs of D. huoshanense, with the advanced methods of isolation and purification, chemical and pharmacological instrument analysis technique of biomacromolecular. Some pharmacological action and mechanisms were investigated. The chemical properties and structural features of main polysaccharides of HPS-1B23 from PLBs of D. huoshanense （HPS） were identified. The main results were obtained as follows:An efficient procedure was developed for PLBs formation from stem segments of D. huoshanense and subsequent subculture for a long time. The highest frequency （79.9%） of PLB formation occurred on MS medium supplemented with 7.5 μmol L^-1 NAA and 0.5 μmol L^-1 KIN. Growth-regulator free MS medium at half-strength was suitable for PLB proliferation. The obtained PLBs were stable in growth, plantlet regeneration, polymorphic DNA of genome and polysaccharide synthesis including content and monosaccharide ratio.Pharmacological studies testified that PLBs have the same potential of synthesizing active polysaccharide as that of wild plants. The crude HPS from wild plants significantly stimulated interferon γ （IFN-γ） release in the supernatant of murine splenocytes and tumor necrosis factor alpha （TNF-α） release in the supernatant of peritoneal macrophages. The critical concentration of HPS was 800 μg mL^-1 for IFN-γ and 200 μg mL^-1 for TNF-α release. RT-PCR analysis indicated those responses were attributed to the up-regulation of the expression of theirown genes in cells. Different HPS fractions showed different capability in stimulating IFN-γ and TNF-α release. By DEAE-Cellulose chromatography, five fractions were all obtained from wild plants and PLBs, in which HPS-1 eluted by distilled water was the main constituent （79%）. These analytical methods of polysaccharide bioactivities could be applied to monitor the liquid culture system of PLBs.The carbon and nitrogen metabolism was analysed and the relationship between PLB growth,更多还原