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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。
Objective To study the differentiation of rat’s stromal cells (MSCs) into neuron-like cells induced by resveratrol in vitro. Methods MSCs were isolated and cultured by whole bone marrow adherent method. MSCs of the third generation were divided into three groups: resveratrol group, control group and normal group. Resveratrol group was given preadiptive solution (DMEM containing 10 μg / L bFGF / F12 medium) for 24 h, then induce the medium (DMEM / F12 medium containing 15 μmol / L resveratrol) for 6 h, then change the maintenance solution (containing 15 μmol / L resveratrol, 10 μg / L bFGF, 2% B27 in DMEM / F12 medium) and continue to culture for 72 h. The control group was the same as the resveratrol induction group. When induced, only DMEM / F12 medium was added for 6 h, The cells were cultured in DMEM / F12 medium containing 10μg / L bFGF and 2% B27 for 72h. The morphological changes of MSCs were observed under inverted microscope. The morphological changes of MSCs were observed at different time points after induction and 2, 6, 24 and 72 h Indirect immunofluorescence, Western blot and RT-PCR were used to detect the expression of nestin and NSE protein and mRNA. Results After resveratrol induction, cell bodies were contracted, protuberant protuberances and neurons were observed. Indirect immunofluorescence staining showed that nestin and NSE were positive in control group and no positive cells in control group. The expression of nestin and NSE protein and mRNA in resveratrol induced group was significantly higher than that in control group, the expression of nestin protein and mRNA reached the peak at 2 h after induction (P <0.01), and then decreased gradually; while NSE protein and mRNA expression gradually increased High, reaching the highest at 72 h after induction (P <0.01), but no significant change in the control group. Conclusion Resveratrol can induce rat bone marrow stromal cells to differentiate into neuron-like cells in vitro, which provides an experimental basis for the application of resveratrol in the field of stem cell transplantation.