目的:研究白花蛇舌草对结肠肿瘤干细胞(CSC)分化的影响,从Wnt信号通路阐明其作用机制。方法:制备白花蛇舌草乙醇提取物,从人结肠肿瘤组织中消化、分拣出结肠CSC。用干细胞球形成试验研究白花蛇舌草对结肠CSC活性的影响,用含胎牛血清的DMEM/F-12培养基诱导CSC分化,并研究白花蛇舌草对结肠CSC分化的影响,以及对结肠CSC中CK20和CD133 mRNA水平的影响,用软琼脂克隆形成试验研究白花蛇舌草对结肠CSC体外转化的影响,用qRT-PCR研究白花蛇舌草对结肠CSC中Wnt信号通路基因β-catenin和TCF4表达的影响。结果:终浓度为125μg/ml、250μg/ml和500μg/ml的白花蛇舌草乙醇提取物可以剂量依赖性地抑制结肠CSC球形成,抑制结肠CSC分化,下调结肠CSC中CK20和CD133 mRNA水平,抑制结肠CSC体外转化,并降低结肠CSC中β-catenin和TCF4 mRNA水平。结论:白花蛇舌草可以抑制结肠CSC分化,其机制在于抑制Wnt信号通路的活性。
Objective: To investigate the effect of Hedyotis diffusa Willd( HDW) on the differentiation of colonic cancer stem cells( CSC) and its underlying mechanism through Wnt signaling pathway. Methods: The ethanol extract of HDW was prepared and the colonic CSC were isolated and harvested from human colonic cancer tissue. The effect of HDW on cell viability of colonic CSC was determined with spheroid formation assay. The effects of HDW on colonic CSC differentiation induced by fetal bovine serum-containing DMEM / F-12 medium and the mRNA level of CK20 and CD133 were explored. The influence of HDW on colonic CSC transformation in vitro was probed with soft agar colony formation assay. The function of HDW on the expression of β-catenin and TCF4,involved in Wnt signaling pathway,in colonic CSC was examined with quantitative real-time RT-PCR. Results: The ethanol extract of HDW at final concentrations of 125 μg / ml,250 μg / ml and 500μg /ml dose-dependently inhibited the spheroid formation and the differentiation of colonic CSC,lowered CK20 and CD133 mRNA level in colonic CSC,suppressed colonic CSC transformation in vitro,and down regulated β-catenin and TCF4 mRNA level in colonic CSC. Conclusions: HDW dampens colonic CSC differentiation by reducing the activity of Wnt signaling pathway.