法舒地尔对C17.2小鼠神经干细胞增殖分化的影响及机制研究  

Effect of fasudil on proliferation and differentiation of neural stem cells and its mechanism in C17.2 mice

作　　者：陈舒[1] 罗铭[2] 刘安民[1] 何明亮[1] 陈美惠[3] 皮荣标[3] 

机构地区：[1]中山大学孙逸仙纪念医院神经外科,广州510120 [2]中山大学孙逸仙纪念医院肿瘤科,广州510120 [3]中山大学药学院药理毒理教研室,广州510006

出　　处：《中华神经医学杂志》2015年第3期244-248,共5页Chinese Journal of Neuromedicine

基　　金：广东省科技社会发展项目（20128031800356）

摘　　要：目的 观察法舒地尔对C17.2小鼠神经干细胞增殖与分化的影响并初步探讨其可能机制. 方法 体外常规培养C17.2细胞,分别加入5、25、50、100 μmol/L法舒地尔作用24h,空白对照组加入等量培养基,噻唑蓝（MTT）法检测细胞存活率,乳酸脱氢酶（LDH）试剂盒检测细胞坏死率的变化;Western blotting检测100 μmol/L法舒地尔组和空白对照组C17.2细胞巢蛋白（nestin）、神经胶质纤维酸性蛋白（GFAP）、双皮质素（DCX）、微管相关蛋白-2（MAP-2）的表达以及Notch信号通路中Notch1、Hes 1蛋白的表达,免疫荧光染色检测C17.2细胞内nestin和GFAP阳性细胞的表达. 结果 与空白对照组比较,50、100 μmol/L法舒地尔组细胞存活率明显降低,且100 μmol/L 法舒地尔组细胞存活率低于50 μmol/L法舒地尔组,差异有统计学意义（P＜0.05）.LDH检测结果显示5组细胞坏死率差异无统计学意义（P＞0.05）.Western blotting检测显示,与空白对照组比较,100μmol/L法舒地尔组细胞nestin表达降低,GFAP、DCX、MAP-2表达增高,Notch信号通路中Notch 1、Hes 1蛋白的表达降低,差异有统计学意义（P＜0.05）.免疫荧光染色显示,与空白对照组比较,100μmol/L法舒地尔组nestin阳性细胞百分比较少,GFAP阳性细胞百分比增加,差异有统计学意义（P＜0.05）. 结论 法舒地尔通过降低Notch信号的表达来抑制C17.2小鼠神经干细胞的增殖,并促使其向神经元和胶质细胞分化.Objective To explore the changes of cell proliferation and differentiation of neural stem cells induced by fasudil treatment,and to primarily study the mechanism in C17.2 mice.Methods C17.2 cells were cultured in vitro; 5,25,50 and 100 μmol/L fasudil were given to the cells,respectively,for 24 h,and cells in the blank control group were given the same volume of culture medium.The changes of cell morphology were observed under a phase-contrast microscope; cell viability and cell necrosis rate were determined by MTT assay and lactate dehydrogenase （LDH） assay,respectively.Western blotting was applied to detect the expression levels of neural markers （nestin,glial fibrillary acidic protein [GFAP],double cortisol [DCX],microtubule-associated protein-2 [MAP-2]）,and Notch Ⅰ and Hes 1 proteins in the notch signaling in cells from the 100 μmol/L fasudil treatment group and blank control group.Immunofluorescence staining was used to detect the nestin and GFAP expressions in the C 17.2 cells.Results As compared with that in the blank control group,the cell viability in the 50 and 100 μmol/L fasudil treatment groups was significantly decreased; that in the 100 μmol/L fasudil treatment group was significantly lower than that in 50 μmol/L fasudil treatment group （P〈0.05）; LDH assay showed no significant difference of cell necrosis among the five groups （P〈0.05）.Western blotting indicated that 100 μmol/L fasudil treatment group had significantly decreased nestin expression,significantly elevated DCX,MAP-2 and GFAP expressions,and statistically decreased expression levels of Notch 1 and Hes 1 as compared with blank control group （P〈0.05）.Immunofluorescence staining indicated that the percentage of nestin positive cells was markedly decreased,the percentage of GFAP positive cells was significantly increased in the 100 μmol/L fasudil treatment group as compared with those in the blank control group （P〈0.05）.Conclusion Fasudil treatment could inhibit the proliferation of C17.2 cells and pro

关 键 词：法舒地尔 C17.2细胞 NOTCH信号通路 细胞增殖 细胞分化 

分 类 号：R965[医药卫生—药理学]