中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2015年
3期
244-248
,共5页
陈舒%罗铭%刘安民%何明亮%陈美惠%皮荣标
陳舒%囉銘%劉安民%何明亮%陳美惠%皮榮標
진서%라명%류안민%하명량%진미혜%피영표
法舒地尔%C17.2细胞%Notch信号通路%细胞增殖%细胞分化
法舒地爾%C17.2細胞%Notch信號通路%細胞增殖%細胞分化
법서지이%C17.2세포%Notch신호통로%세포증식%세포분화
Fasudil%C17.2 cell%Notch signaling pathway%Cell proliferation%Cell differentiation
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.
目的 觀察法舒地爾對C17.2小鼠神經榦細胞增殖與分化的影響併初步探討其可能機製. 方法 體外常規培養C17.2細胞,分彆加入5、25、50、100 μmol/L法舒地爾作用24h,空白對照組加入等量培養基,噻唑藍(MTT)法檢測細胞存活率,乳痠脫氫酶(LDH)試劑盒檢測細胞壞死率的變化;Western blotting檢測100 μmol/L法舒地爾組和空白對照組C17.2細胞巢蛋白(nestin)、神經膠質纖維痠性蛋白(GFAP)、雙皮質素(DCX)、微管相關蛋白-2(MAP-2)的錶達以及Notch信號通路中Notch1、Hes 1蛋白的錶達,免疫熒光染色檢測C17.2細胞內nestin和GFAP暘性細胞的錶達. 結果 與空白對照組比較,50、100 μmol/L法舒地爾組細胞存活率明顯降低,且100 μmol/L 法舒地爾組細胞存活率低于50 μmol/L法舒地爾組,差異有統計學意義(P<0.05).LDH檢測結果顯示5組細胞壞死率差異無統計學意義(P>0.05).Western blotting檢測顯示,與空白對照組比較,100μmol/L法舒地爾組細胞nestin錶達降低,GFAP、DCX、MAP-2錶達增高,Notch信號通路中Notch 1、Hes 1蛋白的錶達降低,差異有統計學意義(P<0.05).免疫熒光染色顯示,與空白對照組比較,100μmol/L法舒地爾組nestin暘性細胞百分比較少,GFAP暘性細胞百分比增加,差異有統計學意義(P<0.05). 結論 法舒地爾通過降低Notch信號的錶達來抑製C17.2小鼠神經榦細胞的增殖,併促使其嚮神經元和膠質細胞分化.
목적 관찰법서지이대C17.2소서신경간세포증식여분화적영향병초보탐토기가능궤제. 방법 체외상규배양C17.2세포,분별가입5、25、50、100 μmol/L법서지이작용24h,공백대조조가입등량배양기,새서람(MTT)법검측세포존활솔,유산탈경매(LDH)시제합검측세포배사솔적변화;Western blotting검측100 μmol/L법서지이조화공백대조조C17.2세포소단백(nestin)、신경효질섬유산성단백(GFAP)、쌍피질소(DCX)、미관상관단백-2(MAP-2)적표체이급Notch신호통로중Notch1、Hes 1단백적표체,면역형광염색검측C17.2세포내nestin화GFAP양성세포적표체. 결과 여공백대조조비교,50、100 μmol/L법서지이조세포존활솔명현강저,차100 μmol/L 법서지이조세포존활솔저우50 μmol/L법서지이조,차이유통계학의의(P<0.05).LDH검측결과현시5조세포배사솔차이무통계학의의(P>0.05).Western blotting검측현시,여공백대조조비교,100μmol/L법서지이조세포nestin표체강저,GFAP、DCX、MAP-2표체증고,Notch신호통로중Notch 1、Hes 1단백적표체강저,차이유통계학의의(P<0.05).면역형광염색현시,여공백대조조비교,100μmol/L법서지이조nestin양성세포백분비교소,GFAP양성세포백분비증가,차이유통계학의의(P<0.05). 결론 법서지이통과강저Notch신호적표체래억제C17.2소서신경간세포적증식,병촉사기향신경원화효질세포분화.
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 promote them differentiate into neuronal and glial cells via decreasing the expression level of Notch signaling.