基于糖芯片的甘肃大宗道地药材中具Aβ42相互作用多糖及寡糖的筛选研究

Carbohydrate microarray is a high-throughput biochip technology developed after the gene chip and protein chip. Based on the technology, the biology function of carbohydrate has been studied in cell adhesion, immune response, tumor metastasis, pathogen infection, etc. through the carbohydrate-protein interactions. It has important applications in glycomics research, clinical testing, and disease diagnosis and drug development. Carbohydrates have a variety of biological functions, and the application of the high-throughput technology to the screening of carbohydrate drugs is the trend of drug development. This study intends to isolate and purify polysaccharides from 10 kinds of staple genuine medicinal materials in Gansu province. The polysaccharides are labeled with fluorescence, and then the polysaccharide microarray is established with nitrocellulose-coated glass sheets as the matrix. Based on the polysaccharide microarray, the interaction between polysaccharide and Alzheimer's disease-related protein Aβ42 is studied to find the active polysaccharide, and the structure of the active polysaccharide is characterized by methylation analysis and nuclear magnetic resonance spectroscopy. The active polysaccharide is degraded by crude acid and the oligosaccharide is separated and prepared on a gel column. The oligosaccharide microarray is established based on the neoglycolipid technology. The interaction of oligosaccharides and Aβ42 is studied by oligosaccharide microarray and the oligosaccharide bound to Aβ42 is found. In addition, the activity of polysaccharides and oligosaccharides discovered by carbohydrate microarray are evaluated on inhibiting Aβ42 aggregation and the toxicity of Aβ42 on nerve cells. A technical system for the discovery of carbohydrate drugs for anti-Alzheimer’s disease is established on the basis of carbohydrate microarray.

糖芯片是继基因芯片和蛋白质芯片之后发展起来的高通量芯片，通过糖与蛋白质的相互作用，研究糖在细胞粘附、免疫应答、肿瘤转移、病原体感染等过程中的功能，在糖组研究、临床检验、疾病诊断和药物开发等领域有重要应用。糖具有多种生物功能，将高通量技术用于糖药物的筛选是药物开发的趋势。本研究拟以甘肃10种大宗道地药材为材料，分离纯化制备多糖，经过荧光标记，以硝酸纤维素薄膜包被的玻璃片为基质构建多糖芯片。基于芯片研究多糖与阿尔兹海默症相关蛋白Aβ42的相互作用发现活性多糖，通过甲基化分析和核磁共振波谱等技术对活性多糖进行结构表征。采用稀酸降解活性多糖，凝胶柱分离制备寡糖，基于拟糖脂技术构建寡糖芯片，通过芯片研究寡糖与Aβ42的相互作用，发现具有与Aβ42结合的寡糖。通过抑制Aβ42聚集和Aβ42对神经细胞毒性的研究，对芯片发现的多糖及寡糖进行活性评价，建立以糖芯片为基础发现抗阿尔兹海默症糖类药物的技术体系。

糖的多种生物学功能不断被发现，并且在医药、食品、化妆品、材料等领域有越来越多的应用。糖分子由于其来源丰富，结构多样，毒副作用小等优势成为药物开发的重要来源，但是由于糖的结构复杂，活性多种多样，如何快速发现活性糖分子并将其进行开发是糖药物研发过程中亟待解决的关键问题之一。糖芯片具有生物芯片高灵敏、高通量等优势，成为糖药物筛选和糖的功能研究的重要手段，近二十多年来广受关注。甘肃省是重要的中药材生产基地，十余种大宗道地药材产量居于全国前列，但对于中药材的深加工以及中药的开发还比较落后，多糖是中药中最重要的活性成分，具有很高的开发利用价值。本研究以甘肃10种大宗道地药材为材料，制备了30余种中药多糖，并经过柱层析获得纯化多糖50余个，在对多糖理化性质和化学结构分析的基础上，进行荧光标记构建了多糖芯片，基于多糖芯片研究发现不同类型果胶与Aβ42具有较好的结合活性，进一步采用酸水解和酶降解、凝胶柱分离制备了不同结构类型的寡糖，质谱分析寡糖的结构序列，并合成了寡糖拟糖脂，基于拟糖脂构建了寡糖芯片，通过芯片研究了寡糖与Aβ42的相互作用，发现具有同型半乳糖醛酸聚糖结构和RGI型果胶来源的寡糖具有更强的结合活性，通过对不同结构寡糖的比较，结合分子对接研究发现，半乳糖醛酸寡糖聚合度越高与Aβ42结合作用越强，同时发现具有支链结构的RGI型甘草来源寡糖能增加与Aβ42形成氢键的机会，且支链为六碳糖比五碳糖形成的作用更强。研究结果为发现具有结构明确，毒副作用小的抗阿尔兹海默症糖药物提供了参考。同时本研究构建的基于糖芯片的活性寡糖发现的技术体系也为糖药物的开发提供了高效的技术平台，为甘肃省大宗道地药材的深入开发和利用奠定了基础。