健脾养胃方对胃癌干细胞基于ABC转运蛋白途径的耐药基因表达和信号传导通路的影响及其药效成分研究

Objective: Based on previous clinical studies from multi-centers with large samples, we aim to exploit the effects of JianpiYangwei（ShenqiJianwei）Decoction on the drug resistance gene expression and signal pathway mediated by ABC transporter protein in CD44＋CD133+ gastric cancer stem cells. Further, to offer a new evidence of traditional Chinese medicine compound for increasing the effectiveness of chemotherapy on gastric cancers, we also aim to unveil the mechanisms of multi-drug resistance of gastric cancer stem cells reversed by JianpiYangwei Decoction and analyze its chemical constituents by establishing the fingerprint , which has great prospects for both clinical application and basic research..Contents: This study is composed of five parts as following: First, we will set up BGC823/5-FU multi-drug resistance gastric cell line by using repeated large dose pulse method combined with drug concentration increment. The characteristic of drug resistance of this cell line will then be evaluated(already finished). Second, we will sort out CD44＋CD133+ gastric cancer stem cells from the drug resistance cell line by fluorescence activated cell sorter (FACS). The stem cell features of those sorted cells will be further identified. Third, we will prepare JianpiYangwei Decoction and control the quality of it by using high performance liquid chromatograph(HPLC). Forth, we will focus on the study of the mechanisms of drug resistance reversal through ABC transporter protein in gastric cancer stem cells by the containing medicine serum of JianpiYangwei Decoction, including the study of changes of drug resistance gene expression and protein such as MDR1, MRP1 and ABCG2, as well as the study of associated signal pathways such as PI3K-AKT, NF–κB, Nrf2, JNK and Wnt/β. Fifth, establish the fingerprint of JianpiYangweiDecoction and analyze its chemical constituents by ultraperformance liquid chromatography-Mass Spectrometry(UPLC-MS) .

研究目的：在前期的大样本多中心临床研究基础上，探讨健脾养胃方（原名参芪健胃汤）含药血清对CD44＋CD133+胃癌干细胞基于ABC转运蛋白途径的耐药基因表达及相关信号传导通路的影响，从干细胞层面阐明健脾养胃方抑制胃癌多药耐药的作用机理，并分析其药效成分及药物归属，为中药复方提高胃癌化疗有效率提供新的依据。.研究内容：一、诱导建立并鉴定胃癌多药耐药细胞系BGC823/5–Fu（已完成）。二、采用磁珠分选术从耐药细胞系中分选培养并鉴定CD44＋CD133+胃癌干细胞。三、制备健脾养胃方并采用HPLC技术对其进行质量控制。四、观察健脾养胃方含药血清对干细胞基于ABC转运蛋白途径的耐药基因MDR1、MRP1、ABCG2 mRNA，及其对相关的信号通路PI3K－Akt、NF–κB、Nrf2、JNK、Wnt/β–连环蛋白的影响。五、采用UPLC-MS技术建立指纹图谱，分析健脾养胃方药效成分及药物归属。