CAJ | 학술논문

目的通过特异性的Racl活性抑制剂下调胶质瘤干细胞(GSCs)中Rac1的活性,探讨Rac1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; Rac1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Rac1活性抑制剂NSC23766处理GSCs,活性实验检测细胞Racl活性,Western blotting检测hMena和基质金属蛋白酶9(MMP-9)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Rac1-三磷酸鸟苷(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(P＜0.05)；与对照组比较,NSC23766处理组GSCs的Rac 1-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(P＜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,Rac1对脑GSCs的迁移和侵袭具有调控作用,抑制Rac1活化可能成为治疗恶性胶质瘤的新策略.
목적 통과특이성적Racl활성억제제하조효질류간세포(GSCs)중Rac1적활성,탐토Rac1재GSCs천이화침습중적작용. 방법 장U251세포치우무혈청DMEM/F12간세포배양기중배양,면역자주분선법분리GSCs,면역형광염색검측CD133감정GSCs; Rac1활성실험검측CD133+여CD133-세포활성,Transwell세포천이화침습실험평개CD133+여CD133-세포적천이화침습능력;가입Rac1활성억제제NSC23766처리GSCs,활성실험검측세포Racl활성,Western blotting검측hMena화기질금속단백매9(MMP-9)단백적표체；Transwell세포천이실험화침습실험평개세포천이화침습능력적개변. 결과 성공배양출현부생장적세포구,가이련속전대병지속표체신경간세포표지물CD133；CD133+세포Rac1-삼린산조감(GTP)표체수평、세포천이화침습적능력현저고우CD133-세포,차이유통계학의의(P＜0.05)；여대조조비교,NSC23766처리조GSCs적Rac 1-GTP、hMena화MMP-9단백표체수평명현강저,천이화침습능력명현감약,차이유통계학의의(P＜0.05). 결론 GSCs상대우종류중적기타세포구유경강적천이화침습능력,Rac1대뇌GSCs적천이화침습구유조공작용,억제Rac1활화가능성위치료악성효질류적신책략.
Objective To explore the role of Rac1 in migration and invasion of glioma stem cells (GSCs) by decreasing the activity of Rac1 with specific Rac 1 inhibitor.Methods U251 cells were cultured in the serum-free DMEM/F12 stem cell medium,and isolation of CD133+ cells (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 Rac1 inhibitor NSC23766 was added into the GSCs; the Rac1 activity was measured by Rac1 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-Rac1 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-Rac1,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; Rac1 modulates the glioma stem cell migration and invasion; inhibition of Rac 1 activation might be a new therapeutic strategy for gliomas.