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前期研究脑表明,脑胶质瘤干细胞(glioma stem cells,GSCs)是胶质瘤发生和发展的重要因素,探索靶向干预GSCs生长有可能成为脑胶质瘤治疗的有效途径之一。该研究旨在阐明两种药物ATRA和γ-分泌酶抑制剂DAPT协同抑制GSCs自我更新的生物学效应。通过用台盼蓝排染法、克隆球形成试验和免疫印迹分析了两种药物的单独使用或联用对GSC样细胞PGC1和PGC2生长、成球能力和自我更新以及干细胞标志物表达的影响。结果发现,单独使用ATRA对PGC1生长有一定的抑制作用,而对PGC2生长几乎没有影响;DAPT对PGCs的生长抑制作用明显强于ATRA。高浓度ATRA(80μmol/L)能诱导PGCs的分化,降低PGCs成球大小,且成球效率降至5%～8%,而正常对照组为32%～35%;同样,DAPT(40μmol/L)也能降低PGCs成球大小,且成球效率降至2%～3%;低浓度ATRA(20μmol/L)和DAPT(5μmol/L)对PGCs自我更新能力和干性没有明显影响,而联合使用后其明显降低PGCs的成球大小,且成球效率降至3%～5%,促进细胞凋亡,并且明显抑制了干细胞标志物Nestin、CD133、Sox2、Oct4的表达,提高了分化标志物GFAP的表达。该研究证明了低浓度的ATRA和DAPT能协同抑制脑胶质瘤干细胞PGCs的自我更新。研究结果将为脑胶质瘤的临床研究提供实验依据。
Previous studies have shown that glioma stem cells (GSCs) are important factors in the development and progression of glioma. To explore the potential role of targeted intervention in the growth of glioma stem cells may be one of the effective ways to treat glioma. This study aimed to elucidate the biological effects of synergistic inhibition of self-renewal of GSCs by ATRA and γ-secretase inhibitor DAPT. The effects of the two drugs alone or in combination on the growth of GSC-like cells PGC1 and PGC2, the ability of spheroidization and self-renewal, and the expression of stem cell markers were analyzed by trypan blue exclusion assay, clone spheroplast assay and immunoblotting. The results showed that ATRA alone inhibited the growth of PGC1 and had little effect on the growth of PGC2. The inhibitory effect of DAPT on PGCs was stronger than that of ATRA. High concentrations of ATRA (80μmol / L) induced the differentiation of PGCs and decreased the size of PGCs into pellets, and the efficiency of pelleting decreased to 5-8% compared with 32% -35% in normal control group. Likewise, DAPT (40μmol / L) ) Also reduced the size of PGCs into pellets and decreased the efficiency of pellet formation to 2% -3%. Low concentrations of ATRA (20μmol / L) and DAPT (5μmol / L) had no significant effect on PGCs self-renewal ability and dryness, After use, it significantly reduced the size of the pellet of PGCs and reduced the efficiency of pellet formation to 3% ~ 5%, promoted cell apoptosis, and markedly inhibited the expression of stem cell markers Nestin, CD133, Sox2 and Oct4 and enhanced differentiation markers GFAP expression. This study demonstrates that low concentrations of ATRA and DAPT synergistically inhibit self-renewal of PGCs in glioma stem cells. The results will provide experimental evidence for the clinical research of glioma.