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。

化疗是胃癌的重要治疗手段，多药耐药(MDR)的存在直接降低了化疗效果，肿瘤干细胞作为肿瘤形成的根源，也是多药耐药的根本原因。健脾养胃方由全国名中医刘沈林教授集多年临床经验创作，依托国家中管局行业专项大样本、多中心课题证实具有降低胃癌术后复发转移的显著优势，并在前期研究中发现其对胃癌耐药具有明确抑制作用。. 本课题通过磁珠分选方法，成功构建胃癌耐药干细胞模型CD44(+)BGC823/5-Fu，并从细胞形态、生长曲线、软琼脂集落形成能力、皮下成瘤能力、干性基因表达情况多方面鉴定了其干细胞特性；健脾养胃方含药血清可从抑制增殖、促进凋亡多方面显著抑制胃癌干细胞耐药性，同时降低了耐药基因P-gp、MDR1、LRP、MRP1、ABCG2的表达，提示本方通过ABC转运蛋白途径发挥对胃癌干细胞耐药的抑制作用；机制研究发现本方对PI3K/Akt、JNK、Nrf2通路主要蛋白及Wnt/β–连环蛋白通路上的CCND1、MYC基因均具有显著抑制作用，有效抑制NF-κB蛋白表达并促进其入核，提示了健脾养胃方逆转干细胞耐药的相关分子转导通路。另外，本研究采用HPLC-Q-TOF-MS/MS技术鉴定得到健脾养胃方中245种化合物，利用TCMSP平台筛选活性成分化合物共41个，主要包括大量有机酸类、黄酮类，另有少量氨基酸类、糖苷类、香豆素类、生物碱等物质，为本方在体内发挥作用的主要药效成分；标准品对照定量测定其中芍药苷、阿魏酸、芦丁、橙皮苷、迷迭香酸、丹酚酸B、甘草酸含量，重复性良好，有效建立了本方的质控标准。. 本课题在前期研究基础上，探讨健脾养胃方对CD44＋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建立质控标准，为中药复方提高胃癌化疗有效率提供新的依据。