金钗石斛多糖抗肿瘤作用的结构基础及其机制研究

Dendrobium nobile Lindl. (Orchidaceae) is a precious traditional Chinese medicine and has been recorded in Chinese Pharmacopoeia (2010 Edition). Its stem is used as a Yin tonic for reinforcing body fluid and nourishing blood. Recently, polysaccharide was demonstrated to be major activity components in D. nobile and shown to possess immuno-stimulating and anti-oxidant activities. Which indicated that polysaccharides from D. nobile maybe have anti-tumor activities since the occurrence and development of tumor were related to redox state and immunity of body tightly. Then we tested the anti-tumor activities of polysaccharides from D. nobile. In our primary study, the water-extracting crude polysaccharides from the stems of D. nobile were shown to exhibit anti-tumor activities in vivo. Further, the crude polysaccharide was submitted to DEAE-cellulose anionexchange and gel permeation chromatography to afford a homogeneous polysaccharide DNP-W1A, which exhibited high inhibition ratios of 68% and 84% against the growth of Sarcoma 180 tumor cells in vivo and HL-60 cells in vitro, respectively. Which suggested that DNP-W1A had the potential for development for antitumor therapy. On the base of the above results, we carried out further study on DNP-W1A in the present work. The research contents include three parts. One is that the structural features of DNP-W1A, including primary and secondary structure, were charactered with the combination of chemical and physical techniques. The other is that the structure-activities relationship of DNP-W1A was determined by enzymatic method. The last is that the anti-tumor mechanism was elucidated through finding out the effects of DNP-W1A on Bcl-2 signal pathway during tumor cell apoptosis. Briefly, we hope to clarify chemical structure of anti-tumor activities of polysaccharide DNP-W1A and together with the primary biological mechanism. This work provides valuable and fundamental information to further develop antitumor medicine and functional food using polysaccharide and understand the use of traditional Chinese medicine.

金钗石斛为中国药典记载的名贵中药，具有'除痹下气，补五脏虚劳籯瘦，强阴滋精'之功效。在前期工作中，我们发现金钗石斛主要活性化学成分多糖具有较高的抗肿瘤活性，并从中筛选出了一个高活性的抗肿瘤多糖组分DNP-W1A，其对小鼠体内Sarcoma 180肉瘤增殖的抑制率大于68%，对人早幼粒细胞白血病细胞株HL-60增殖的抑制率大于84%，预示了DNP-W1A具有开发成抗肿瘤新药的良好前景。本课题即是在前期工作基础上，对DNP-W1A进行进一步的研究，内容包括采用化学、物理与光谱学手段，并结合高分子溶液理论表征DNP-W1A的一级结构和高级结构；采用酶法修饰的手段解析DNP-W1A的抗肿瘤活性构效关系；通过了解DNP-W1A对肿瘤细胞凋亡过程中Bc1-2信号途径的影响，探讨DNP-W1A的抗肿瘤作用机理。课题的完成为定向开发多糖类抗肿瘤活性新药及中国传统名贵中药的开发利用提供基础数据和科学依据。

金钗石斛（Dendrobium nobile Lindl.）是中国药典（2010版）收载的名贵中药，多糖是其主要活性成分。课题采用热水浸提法获得金钗石斛水提粗多糖DNP-W，并通过DEAE-纤维素和葡聚糖凝胶对DNP-W进行分离纯化，获得了主要的均一抗肿瘤活性多糖组分DNP-W1A。一级结构表征结果表明DNP-W1A主要由甘露糖组成的半乳葡萄甘露聚糖，分子量为2.4 ×104，比旋光度[α]D20为+17.1°（c 2.5），由甘露糖、葡萄糖和半乳糖三种单糖组成，摩尔比为5.4:1.6:1.0。DNP-W1A的主链由1,6-linked Manp和1,3-linked Manp组成，其中1,6-linked Manp的2位和3位存在分支。2位上连接的支链由1,6-linked Galp和1-linked Galp组成, 3位上连接的支链由1,4-linked Glcp和1-linked Glcp组成；DNP-W1A的高级结构分析表明DNP-W1A不具有完整单晶结构，其玻璃化转变温度为85℃，在溶液中主要以半柔顺链的无规线团存在。. 课题进一步采用硫酸化分子修饰和酶解构建了DNP-W1A抗肿瘤活性构效关系。利用氯磺酸-吡啶法和酶解法分别获得9个DNP-W1A硫酸酯化多糖片段和3个DNP-W1A酶解片段。抗肿瘤活性实验结果表明多糖DNP-W1A及其衍生物都有较高的抗肿瘤活性，但不同多糖片段之间活性差异较大，硫酸化后的多糖片段活性与原糖相比活性得到显著提高，且与取代度和分子量有关。构效关系分析表明DNP-W1A的抗肿瘤活性与单糖组成、支链、分子量及其连接方式等都有关，特别是可能与其高含量的甘露糖含量有关。. 课题最后采用Annexin-FITC/PI流式细胞仪、荧光定量RT-PCR和Western Blot等技术手段探讨了DNP-W1A抗肿瘤分子作用机理。结果表明DNP-W1A抗肿瘤作用主要是通过促进肿瘤细胞凋亡，上调Bax、Caspase-3和Caspase-9等基因的表达量，下调Bcl-2基因表达量及P-AKT蛋白合成实现的。. 课题的完成为金钗石斛及其多糖的深入研究提供了大量有价值的基础数据，也为中国传统名贵中药的开发利用提供了科学依据，具有学术价值。