CAJ | 학술논문

目的探讨核转录因子-κB(NF-κB)信号通路在法舒地尔诱导大鼠骨髓间充质干细胞(MSCs)向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(含终浓度200 μmol/L法舒地尔的DMEM培养基900 μL+10％胎牛血清100 μL),C组加诱导液(含终浓度200 μmol/L法舒地尔和1000μg/L脂多糖的DMEM培养基900 μL+10％胎牛血清100 μL);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(NSE)、神经元微管结合蛋白(MAP-2)、细胞周期蛋白D1(cyclin D1)和胶质纤维酸性蛋白(GFAP)的表达,采用实时定量PCR(RT-PCR)检测cyclin D1 mRNA及MAP-2 mRNA的表达,采用Westem blotting检测3组细胞cyclin D1及MAP-2蛋白的表达.结果 (1)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,cyclin D1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Western blotting结果显示,与A组比较,B组及C组的cyclin D1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(P＜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.
목적 탐토핵전록인자-κB(NF-κB)신호통로재법서지이유도대서골수간충질간세포(MSCs)향신경원분화중적작용.방법 취배양지소4대이상적대서MSCs,분위무혈청배양기대조조(A조)、법서지이조(B조)급법서지이+지다당조(C조);A조지가무혈청DMEM배양기,B조가유도액(함종농도200 μmol/L법서지이적DMEM배양기900 μL+10％태우혈청100 μL),C조가유도액(함종농도200 μmol/L법서지이화1000μg/L지다당적DMEM배양기900 μL+10％태우혈청100 μL);분별유도6h.수후채용도치형광현미경관찰각조세포형태변화,채용세포형광면역조화법검측신경원특이성희순화매(NSE)、신경원미관결합단백(MAP-2)、세포주기단백D1(cyclin D1)화효질섬유산성단백(GFAP)적표체,채용실시정량PCR(RT-PCR)검측cyclin D1 mRNA급MAP-2 mRNA적표체,채용Westem blotting검측3조세포cyclin D1급MAP-2단백적표체.결과 (1)A조MSCs기본보지편평、돌기희소,B조급C조가견신경원양세포명현증다,형성전형신경망락.기중B조MSCs향신경원분화속도쾌,C조MSCs향신경원분화속도교만,신경원망락교B조희소.(2)형광면역조화결과현시,여A조비교,B조급C조NSE여MAP-2표체량균명현증가,cyclin D1표체균명현하강,차이균유통계학의의(P＜0.05).3조세포적GFAP표체솔균소우1％,차이무통계학의의(P＞0.05).(3)RT-PCR여Western blotting결과현시,여A조비교,B조급C조적cyclin D1단백급mRNA표체균명현감소,MAP-2단백급mRNA표체균명현증가,차이균유통계학의의(P＜0.05).결론 NF-kB신호통로통과조절cyclinD1표체삼여법서지이유도대서MSCs향신경원분화적작용.
Objective To investigate the effect of nuclear-transcription factor-κB 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-free 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 of fasudil),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 mRNA expressions ofcyclin 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 expressions and significantly higher MAP-2 protein and mRNA expressions (P＜0.05).Conclusion The NF-κB signal transduction pathway,by regulating the expression of cyclin D1,participates in the neuronal differentiation of rat marrow mesenchymal stem cells induced by fasudil.