摘要
目的通过特异性的Racl活性抑制剂下调胶质瘤干细胞(GSCs)中Racl的活性,探讨Racl在GSCs迁移和侵袭中的作用。方法将U251细胞置于无血清DMEM/F12干细胞培养基中培养,免疫磁珠分选法分离GSCs,免疫荧光染色检测CD133鉴定GSCs;Racl活性实验检测CD133+与CD133-细胞活性,Transwell细胞迁移和侵袭实验评价CD133+与CD133-细胞的迁移和侵袭能力;加入Racl活性抑制剂NSC23766处理GSCs,活性实验检测细胞Racl活性,Westernblotting检测hMena和基质金属蛋白酶9(MMP-91蛋白的表达;Transwell细胞迁移实验和侵袭实验评价细胞迁移和侵袭能力的改变。结果成功培养出悬浮生长的细胞球,可以连续传代并持续表达神经干细胞标志物CD133;CD133+细胞Racl-三磷酸鸟苷(GTP)表达水平、细胞迁移和侵袭的能力显著高于CD133-细胞,差异有统计学意义伴0.05);与对照组比较,NSC23766处理组GSCs的Racl-GTP、hMena和MMP-9蛋白表达水平明显降低,迁移和侵袭能力明显减弱,差异有统计学意义fP〈0.05)。结论GSCs相对于肿瘤中的其他细胞具有更强的迁移和侵袭能力,Racl对脑GSCs的迁移和侵袭具有调控作用,抑制Racl活化可能成为治疗恶性胶质瘤的新策略。
Objective To explore the role of Racl in migration and invasion of glioma stem cells (GSCs) by decreasing the activity of Racl with specific Racl inhibitor. Methods U251 cells were cultured in the serum-free DMEM/FI2 stem cell medium, and isolation of CD133+ ceils (GSCs) from U251 cells was performed; immunofluorescence was conducted to identify GSCs. Racl activity assay was employed to detect the activity of CD133+ and CD133- cells; the migration and invasion of cells were detected by Transwell cell migration/invasion assay. Specific Racl inhibitor NSC23766 was added into the GSCs; the Racl activity was measured by Racl activation assay and Western blotting was conducted to detect the expressions of human orthology of mammalian enabled (hMena) and matrix metalloproteinase- 9 (MMP-9). Transwell cell migration/invasion assay was employed to detect the migration and invasion of these cells. Results GCSs formed cell sphere, floating in the medium; these cells could continuous passage and persistently express the stem cell marker CD133. The GTP-Racl expression and migration/invasion of CD133+ cells were significantly higher than those of CD133- cells (P〈0.05). In comparison with those in the control group, the expressions of GTP-Racl, hMena and MMP-9 in NSC23766 treatment group were significantly decreased (P〈0.05). Conclusion GSCs enjoy stronger migration and invasion abilities than other tumor cells; Racl modulates the glioma stem cell migration and invasion; inhibition of Rac 1 activation might be a new theraoeutic strategy for gliomas.
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2013年第3期221-225,共5页
Chinese Journal of Neuromedicine
基金
国家自然科学基金(30772228)