白藜芦醇体外诱导大鼠骨髓基质细胞向神经元样细胞的分化

目的研究白藜芦醇体外诱导大鼠骨髓基质细胞(Marrow stroma cells,MSCs)向神经元样细胞的分化。方法采用全骨髓贴壁法分离培养MSCs,取第3代MSCs分为白藜芦醇诱导组、对照组和正常组,白藜芦醇诱导组先加入预诱导液(含10μg/L bFGF的DMEM/F12培养基)培养24 h,再更换诱导液(含15μmol/L白藜芦醇的DMEM/F12培养基)诱导6 h,然后更换维持液(含15μmol/L白藜芦醇,10μg/L bFGF,2%B27的DMEM/F12培养基),继续培养至72 h;对照组预诱导与白藜芦醇诱导组相同,诱导时仅加入DMEM/F12培养基诱导6 h,再更换维持液(含10μg/L bFGF,2%B27的DMEM/F12培养基),继续培养至72 h,倒置显微镜下观察诱导分化后MSCs形态;各组分别于诱导前及诱导后2、6、24、72 h采用间接免疫荧光法、Western blot法和RT-PCR法检测nestin、NSE蛋白及mRNA表达。结果白藜芦醇诱导后细胞胞体收缩,伸出长突起,类似神经元,间接免疫荧光染色显示诱导后细胞nestin和NSE阳性,对照组未见阳性细胞。白藜芦醇诱导组nestin、NSE蛋白及mRNA表达较对照组明显升高,诱导后2 h,nestin蛋白及mRNA表达达最高(P<0.01),之后逐渐下降;而NSE蛋白及mRNA表达逐渐升高,诱导后72 h达最高(P<0.01),对照组则无明显变化。结论白藜芦醇在体外可诱导大鼠骨髓基质细胞分化为神经元样细胞,为白藜芦醇在干细胞移植领域的应用提供了实验依据。

Differentiation of rat marrow stroma cells into neuron-like cells induced by resveratrol in vitro

Objective To investigate the differentiation of rat bone marrow stromal cells(MSCs) into neuron-like cells induced by resveratrol in vitro.Methods MSCs were isolated from the whole bone marrow of rats by adherent culture in vitro.The MSCs of passage 3 were divided into resveratrol,control and normal groups.The MSCs were pre-induced with DMEM / F12 containing 10 μg / L bFGF for 24 h,and induced with DMEM / F12 containing 15 μmol / L resveratrol for 6 h,then added with maintain medium(DMEM / F12 containing 15 μmol / L resveratrol,10 μg / L bFGF and 2% B27) and further cultured until 72 h.The MSCs in control group were pre-induced by the same method,the induced with DMEM / F12 for 6 h,added with maintain medium(DMEM / F12 containing 10 μg / L bFGF and 2% B27) and further cultured until 72 h.However,the MSCs in normal group were untreated.The MSCs were observed for morphology under invert microscope 72 h after induction,in which the expression levels of nestin and NSE proteins and mRNAs were determined by indirect IFA,Western blot and RT-PCR before and 2,6,24 and 72 h after induction.Results The MSCs after induction with resveratrol were shrunk,on which neuron-like enations were observed.IFA showed that the MSCs after induction were positive for nestin and NSE,while those in control group were negative.The expression levels of nestin and NSE proteins and mRNAs in MSCs induced with resveratrol were significantly higher than those in control group.The expression levels of nestin protein and mRNA reached the peak values 2 h after induction(P < 0.01) then decreased gradually.However,the expression levels of NSE protein and mRNA increased gradually and reached the peak values 72 h after induction(P < 0.01).The expression levels in control group showed no significant change.Conclusion Resveratrol induced the differentiation of MSCs into neuron-like cells in vitro,which provided an experimental basis for application of resveratrol to stem cell transplantation.