枸杞多糖抑制肿瘤代谢靶标分子的分离富集及其代谢机制研究

Screening and discovery of new effective biomarkers in the process of Lycium barbarum polysaccharides inhibit tumor metabolism in serum, which is of great significance for molecular mechanism and clinical outcome prediction. The objective of this project is to prepare a series of bio-affinity materials with high selectivity and super-large specific surface for capture, separation and to identify the target molecule-endogenous phosphopeptides with high sensitivity, which are biomarkers from Lycium barbarum polysaccharides to induce the apoptosis of tumor cells with differential expression of phosphorylated peptides produced by hydrolysis and degradation of phosphorylated proteins. Using layer-by-layer assembly method, a facile synthetic approach was proposed for the novel magnetic networks affinity materials by Lignin-assisted exfoliation of molybdenum disulfide as carrier and polydopaminelayer modified composite metal ions and metal oxide as functional assistant. The structure-activity relationship between affinity materials and target molecular or protein digestion solution, and the effects on the enrichment selectivity, adsorption capacity, and the improvement of phosphopeptide mass spectrometry were systematically investigated. Combining with bio-mass spectrometry technology, the molecular mechanism of different expressed phosphorylated peptides for Lycium barbarum polysaccharides anti-tumor metabolism was explored on the molecular level using mouse model. The PCA-LDA classification mathematical model for fingerprint spectrum of accurate identification of highly specific, highly sensitive biological target metabolism was established. It is significant to expand and enrich the composition of blood proteome and to study the molecular mechanism of plant polysaccharides inhibiting tumor metabolism.

筛选和发现枸杞多糖抑制肿瘤代谢过程血清中新的有效生物标志物，对于分子作用机制及临床结果预测具有重要意义。项目以合成高选择性、超大比表面的生物亲和材料，用于分离、富集且高灵敏度地鉴定枸杞多糖抑制肿瘤代谢的靶标分子-内源性磷酸肽段为主要目标，以木质素插层改性的网络状二硫化物为载体、聚多巴胺修饰复合金属离子或金属氧化物为功能化助剂，采用层层组装方法制备系列磁性网络结构材料，并探究该类材料对生物靶标分子内源性磷酸肽段、蛋白酶切液等在富集选择性、吸附容量及改善磷酸肽质谱信号等方面的变化规律、作用机理和构效关系，继而应用生物质谱技术等从分子层面研究磷酸化肽段的差异表达，探讨枸杞多糖抗肿瘤代谢内在的分子机制，建立能够准确区分具有高特异性、高灵敏的生物临床靶标物代谢指纹谱的PCA-LDA分类数学模型等。项目研究对于拓展和丰富血液蛋白质组的组成及植物多糖抑制肿瘤分子代谢机制的研究具有重要意义。

研究枸杞多糖抑制肿瘤代谢过程血清中磷酸化肽蛋白组表达的差异，对探讨分子作用机制及临床结果预测具有重要意义。该项目以二硫化钼（MoS2）为基体，以TiO2和ZrO2纳米粒子作为捕获磷酸化肽的亲和探针，构建TiO2@mag-MoS2/石墨烯、ZrO2@mag-MoS2/石墨烯和TiO2/ZrO2@MoS2高效亲和吸附材料。通过研究亲和探针对生物靶标分子内源性磷酸化肽的选择性吸附作用机理和构效关系，实现了高选择性分离、富集蛋白质酶切液中内源性磷酸化肽段。以非小细胞肺癌细胞系A549为载体，结合选择性亲和探针的分离富集、灵敏的生物质谱(HPLC-MSn)鉴定和生物信息学方法的解析。我们开展了枸杞多糖(LBP)作用于A549细胞的体外实验和体内实验，探究了枸杞多糖(LBP)促进A549细胞的细胞凋亡及凋亡相关蛋白表达的影响。体外实验结果显示，一定浓度的枸杞多糖(LBP)能显著抑制细胞的增殖，抑制率大于30%，同时促进人肺癌A549细胞的凋亡，促进了凋亡因子Bax的表达上调和抑制了凋亡因子Bcl-2的表达下调。其作用机制可能源于PTK2、MAPK、EIF5B、RB1和ULK1等关键凋亡蛋白质在多方面生物功能和多条信号通路中发挥的作用。小鼠体内成瘤实验结果显示，枸杞多糖对小鼠体内肿瘤的抑制不显著，可能的原因是LBP在体内吸收代谢机制的复杂性导致。该项目具有系统的实验方案和可靠的数据，为拓展植物多糖影响肿瘤代谢的机制研究和临床预测提供了理论依据。