马齿苋醇提取物对结肠癌细胞及其干细胞体外增殖作用的机理研究

Effect of ethanol extract from Machixian on colon cancer cells and their stem cell proliferation in vitro: a mechanism study

目的探讨马齿苋醇提取物对结肠癌细胞及其干细胞体外增殖能力的影响及其作用机制。方法对结肠癌干细胞进行无血清培养,CCK-8法测定不同浓度马齿苋醇提取物对结肠癌HT-29细胞及HT-29干细胞的生长抑制情况,流式细胞仪检测其对HT-29细胞及其干细胞的凋亡情况及对细胞周期的影响。RT-PCR法以及Western blot分析马齿苋醇提取物对HT-29细胞及其干细胞Notch、Wnt双信号通路中Notch1、Notch2及β-catenin基因及蛋白表达的影响。结果马齿苋醇提取物对HT-29细胞及HT-29干细胞增殖有抑制作用,其作用时间、剂量与细胞增殖抑制率呈正相关,且对HT-29细胞增殖的抑制作用及凋亡率的影响较HT-29干细胞显著(P<0.05)。马齿苋醇提取物可下调HT-29细胞及其干细胞Notch1、β-catenin基因表达,并下调Notch1、Notch2及β-catenin蛋白表达(P<0.05)。结论马齿苋醇提取物可以通过下调Notch-1、Notch-2、β-catenin蛋白的表达发挥体外抑制结肠癌细胞及其干细胞增殖的作用。

Objective To discuss the influence of ethanol extract from Machixian （Herba Portulacae, Portulaca） on colon cancer cells and their stem cell proliferation in vitro and effective mechanism. Methods The stem cells of colon cancer were cultivated with serum free medium. The inhibitory effects of ethanol extract from Machixian in difference doses on growth of HT-29 cells and HT-29 stem cells were detected by using CCK-8 assay. The influence of ethanol extract from Machixian on apoptosis and cell cycle of HT-29 cells and HT-29 stem cells was detected by using flow cytometer. The influence of ethanol extract from Machixian on genetic and protein expressions of Notchl, Notch2 and β-catenin in Notch signal pathway and Writ signal pathway of HT-29 cells and HT-29 stem cells was analyzed by using reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting method. Results The ethanol extract from Machixian had inhibitory effect on proliferation of HT-29 cells and HT-29 stem cells,its effective time and dose were positively correlated to cellular proliferation inhibition rate, and its inhibitory effect on proliferation and improve effect on apoptosis rate were more significant on HT-29 cells than those on HT-29 stem cells （ P 〈 0.05 ）. The ethanol extract from Machixian could down-regulate genetic expressions of Notchl and β-catenin and protein expressions of Notchl, Notch2 and β-catenin of HT-29 cells and HT-29 stem cells （ P 〈 0.05 ）. Conclusion The ethanol extract from Machixian can inhibit in vitro the proliferation of colon cancer cells and their stem cells through down-regulating protein expressions of Notchl, Notch2 and β-catenin.