固定化FGFR4高表达细胞色谱新方法的建立及在 “七药”活性成分分析中的应用

Cell membrane chromatography (CMC) was found in 1996 and was been wide application and well evaluated. However, the applicant study the theory and develop new method on CMC since 1997, and found that CMC is difficult to distinguish the inner or outer surface of the cell membrane. CMC cannot reflect the real interaction between ligand and cell, and the further application of CMC is limited. On the basis of the theory of CMC, we put forward a new biological affinity chromatography conception – immobilized cell chromatography (CC), and the technique and method of cell biology, chromatography and in vitro pharmacology will be used. In this project, the FGFR4 over expressed cell line is cultured on micro carrier. Then, immobilized cell stationary phase will be prepared using modified immunocytochemistry cell fixation technique. Using the stationary phase, the cell chromatography will established. To improve the identification and qualification of cell chromatography for the screening efficiency, the cell chromatography will be hyphenated with LC/MS to established a two dimensional CC-LC/MS system. The system will be used to screen the active components from the “Qiyao” a kind of folk herb medicine in “Qin-Ba” mountain area. The screened compound will be test by the method of cell biology, and in vitro pharmacology and ensure their effective. The establish of CC method and the results of the project will provide a new methodological basis for material study of traditional Chinese medicine, and fundamental basis for higher efficiency screening method.

细胞膜色谱法（CMC）已得到广泛应用和良好评价。但其细胞膜固定相无法只反映细胞膜外表面特性，会造成药物与细胞膜受体作用强度的测定偏差。针对该问题，本项目在CMC基础之上提出新的生物亲和色谱构想-固定化细胞色谱法。应用细胞生物学技术和色谱技术结合离体药理学实验方法，利用高表达FGFR4细胞结合微载体培养技术，以免疫细胞化学细胞固定技术制备固定化细胞固定相；建立固定化高表达FGFR4细胞色谱法，用于模拟药物与细胞的相互作用。以其为核心技术与LC-MS结合建立在线二维分析系统，提高活性筛选的定性与定量水平。并应用于秦巴山区特有药用植物“七药”中可特异性作用于高表达FGFR4细胞的活性成分筛选；结合分子生物学、离体药理学等技术对活性成分作用进行验证。项目的完成将丰富生物亲和色谱内容，拓展色谱技术在药物分析中的应用；为中药活性成分筛选分析提供新技术和方法。

细胞膜色谱法（CMC）已得到广泛应用和良好评价。但其细胞膜固定相无法只反映细胞膜外表面特性，会造成药物与细胞膜受体作用强度的测定偏差。针对该问题，本项目在CMC基础之上提出新的生物亲和色谱构想-固定化细胞色谱法。通过优化培养方法利用微载体培养高表达HEK293-FGFR4细胞，研究了多聚甲醛固定方法，从而完成细胞固定相的制备，电镜结果显示HEK293-FGFR4细胞单层分布于cytodex 3表面，细胞可保持基本形态；应用扫描电镜、DAPI染色和场发射能谱扫描对固定相细胞进行表征，结果表明湿法装柱对固定相无影响；通过对流动相、pH值、柱温等条件考察，得到优化的色谱条件为：2mmol/L磷酸盐，pH为7.4，柱温25℃；通过色谱方法学研究，表明建立的高表达HEK293-FGFR4细胞色谱可以特异性的筛选靶向FGFR4的药物；建立了5种FGFR4抑制剂的RP-HPLC快速分析方法，测定其与细胞色谱的结合能力，结果表明结合能力PD173074>AZD4547>BGJ398>BIBF1120>Gefitinib；建立了细胞色谱区带流出模型，测定了PD173074、AZD4547、BGJ398、BIBF1120、Gefitinib的平衡解离常数，并发现PD173074与AZD4547、BGJ398、BIBF1120竞争相同的作用位点，而与Gefitinib的作用位点不同；通过建立HEK293-FGFR4/CC-online-HPLC/MS二维在线联用方法，从白花前胡中“识别”-“分析”-“鉴定”出能够与FGFR4相互作用的活性组分白花前胡甲素；利用受体配体对接实验表明白花前胡甲素和FGFR4的作用位点与PD173074和FGFR4的作用位点相同；细胞增殖抑制实验发现，白花前胡甲素对HEK293-FGFR4细胞的抑制具有浓度依赖性。同时，利用建立的细胞色谱方法应用于吴茱萸活性成分筛选，联合HPLC-MS鉴定出吴茱萸碱和柠檬苦素为活性组分；分子生物实验结果表明其对细胞增殖活性的抑制作用。项目的完成不仅丰富了生物亲和色谱内容，拓展色谱技术在药物分析中的应用，也为中药活性成分筛选分析提供了新技术和方法。