黄芪多糖抑制黑色素瘤干细胞自我更新的实验研究

Experimental Study on Astragalus Polysaccharide Inhibiting Self-renewal of Melanoma Stem Cells

目的观察黄芪多糖对黑色素瘤干细胞自我更新的作用,并探究了其可能的分子机制。方法采用无血清培养的方法从人源A375及鼠源B16F10黑色素瘤细胞中诱导黑色素瘤干细胞,建立黑色素瘤干细胞体外培养体系,通过二次球落形成实验探讨黄芪多糖对黑色素瘤干细胞自我更新的作用,荧光定量PCR及蛋白质免疫印迹法检测黄芪多糖对黑色素瘤干细胞表面标志物ALDH1A3及核转录因子SOX2、OCT4、Nanog在mRNA及蛋白水平的表达情况。结果在无血清培养法培养的黑色素瘤干细胞球落中,黑色素瘤干细胞表面标志物ALDH1A3及核转录因子SOX2、OCT4、Nanog在mRNA及蛋白水平表达增高,表明本研究成功的建立了黑色素瘤干细胞的体外培养体系。用黄芪多糖作用于黑色素瘤干细胞后,二次球落形成实验发现黄芪多糖可以抑制黑色素瘤干细胞的球落形成数量及体积。荧光定量PCR及蛋白质免疫印迹法显示:与对照组相比,黄芪多糖可降低黑色素瘤干细胞表面标志物ALDH1A3及核转录因子SOX2、OCT4、Nanog在mRNA及蛋白水平的表达。结论黄芪多糖可抑制黑色素瘤干细胞的自我更新,其作用机制可能与下调ALDH1A3、SOX2、OCT4、Nanog的表达有关。

Objective To investigate the effect of Astragalus polysaccharide on the self-renewal of melanoma stem cells,and its underlying molecular mechanism.Methods Melanoma stem cells were induced from A375 and B16 F10 melanoma cells by serum-free medium culture.The melanoma stem cells were treated with Astragalus polysaccharide.The effect of Astragalus polysaccharide on melanoma stem cell self-renewal ability was detected by secondary sphere formation assay.And the expressions of ALDH1 A3 and nuclear transcription factors SOX2,OCT4 and Nanog were detected by real-time fluorescent quantitative PCR and Western blotting.Results The result showed that the mRNA and protein expression levels of ALDH1 A3 and nuclear transcription factors SOX2,OCT4 and Nanog in melanoma stem cells were also higher than those in serum-containing medium group.This indicated that the culture system of melanoma stem cells was successfully established in this study.Then melanoma stem cells were treated with Astragalus polysaccharide.The secondary sphere formation assay showed that Astragalus polysaccharide could inhibit the number and volume of tumorspheres of melanoma stem cells.The mRNA and protein expression levels of ALDH1 A3 and nuclear transcription factors SOX2,OCT4 and Nanog were lower than those in the control.Conclusion The results indicated Astragalus polysaccharide can inhibit the self-renewal of melanoma stem cells.The mechanism may be related to the down-regulation of ALDH1 A3,SOX2,OCT4 and Nanog.