NF-kB信号通路在法舒地尔诱导大鼠骨髓间充质干细胞向神经元分化中的作用  

Nuclear-transcription factor-KB signal transduction pathway participating in neuronal differentiation of rat marrow mesenchymal stem cells induced by fasudil

作　　者：赵二义[1] 贾延劼[3] 王带媚[1] 文国强[1] 景黎君[3] 邓益东[1] 郭敏[2] 

机构地区：[1]海南省人民医院神经内科,海口570311 [2]海南省人民医院科教处,海口570311 [3]郑州大学第一附属医院神经内科,郑州450052

出　　处：《中华神经医学杂志》2015年第9期899-904,共6页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81260209）;海南省重点科技项目（Zdxm20130073）;海南省社会发展科技专项（SF201404）;海南省自然科学基金（813208）

摘　　要：目的探讨核转录因子-kB（NF-kB）信号通路在法舒地尔诱导大鼠骨髓间充质干细胞（MSCs）神经元分化中的作用。方法取培养至少4代以上的大鼠MSCs，分为无血清培养基对照组（A组）、法舒地尔组（B组）及法舒地尔＋脂多糖组（C组）；A组只加无血清DMEM培养基，B组加诱导液f含终浓度200μmol／L法舒地尔的DMEM培养基900μL＋10％胎牛血清100μL），C组加诱导液（含终浓度200μmol／L法舒地尔和1000μg／L脂多糖的DMEM培养基900μL＋10％胎牛血清100μL）：分别诱导6h。随后采用倒置荧光显微镜观察各组细胞形态变化，采用细胞荧光免疫组化法检测神经元特异性烯醇化酶州SE）、神经元微管结合蛋白（MAP-2）、细胞周期蛋白D1（cyclinD1）和胶质纤维酸性蛋白（GFAP）的表达．采用实时定量PCR（RT—PCR）检测cyclinD1mRNA及MAP-2 mRNA的表达，采用Westernblotting检测3组细胞cyclinD1及MAP-2蛋白的表达。结果f1）A组MSCs基本保持扁平、突起稀少，B组及C组可见神经元样细胞明显增多，形成典型神经网络。其中B组MSCs向神经元分化速度快，C组MSCs向神经元分化速度较慢，神经元网络较B组稀少。（2）荧光免疫组化结果显示，与A组比较，B组及C组NSE与MAP-2表达量均明显增加，cyclinDl表达均明显下降，差异均有统计学意义（P〈0．05）。3组细胞的GFAP表达率均小于1％，差异无统计学意义（P〉0．05）。（3）RT-PCR与Westernblotting结果显示，与A组比较，B组及C组的cyclinDl蛋白及mRNA表达均明显减少，MAP-2蛋白及mRNA表达均明显增加．差异均有统计学意义（JD〈0．05）。结论NF—kB信号通路通过调节cyclinD1表达参与法舒地尔诱导大鼠MSCs向神经元分化的作用。Objective To investigate the effect of nuclear-transcription factor-KB signal transduction pathway on differentiation of rat marrow mesenchymal stem cells （MSCs） into neurons. Methods The MSCs were purified at least four generations in vitro. And then, the MSCs were assigned into three groups： non-serum control group （group A）, fasudil treatment group （group B） and fasudil and lipopolysaccharide treatment group （group C）. Cells in group A were added serum-flee DMEM, those in group B were added induced liquid （DMEM 900 μL＋fetal bovine serum 100 μL that contains final concentration of 200 μmol/L offasudil）, and those in group C were added induced liquid （DMEM 900 μL＋fetal bovine serum 100 μL that contains final concentration of 200 μmol/1 of fasudil and final concentration of 1000 μg/L of lipopolysaccharide）. All the MSCs were induced into neurons for 6 h. The morphology of MSCs was observed under inverted fluorescence microscope. The expressions of neuronspecific enolase （NSE）, microtubule-associated protein （MAP）-2, cyclin D1 and glial fibrillary acidic protein （GFAP） in neurons were detected by immunocytochemical method. The rnRNA expressions of cyclin D1 and MA P-2 in neurons were detected by real time-PCR. The protein expressions ofcyclin D1 and MAP-2 were detected by Western blotting. Results （1） The MSCs of group A kept flat and the neurites were scarce; the MSCs of group B and C could differentiate into neurons, and the speed of MSCs of group B differentiating into neurons was faster than that in group C; there was typical neural network in group B and the neural network in group C was scarcer than group B. （2） As compared with those in group A, higher expression levels of NSE and MAP-2, and lower cyclin D1 expression level in group B and group C were noted （P〈0.05）; the expression percentage of GFAP was lower than 1% in both three groups. （3） As compared with group A, group B and group C had significantly lower cyclin D1 protein and mRNA e

关 键 词：核转录因子-KB 法舒地尔 骨髓间充质干细胞 神经元 细胞分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]