hBMSCs向神经细胞分化前后表达神经生长因子变化的实验研究

目的:检测人骨髓间充质干细胞(hBMSCs)向神经细胞诱导分化前后培养液上清中神经生长因子(NGF)含量的变化,探讨BMSCs移植作用机制。方法:从健康志原者髂骨处抽取骨髓5 mL,体外分离、培养、扩增后用400 ng/L音猬分子(SHH)、0.5μmol维甲酸(RA)和5μmol Forskolin(Fn)组成的诱导体系进行诱导。使用ELISA法检测1 d,3 d,5 d,7 d的hBMSCs培养液上清中NGF的浓度,并且对比诱导前(12 h,1 d,3 d)后(12 h,1 d,3 d)的上清液中该物质浓度的变化。结果:hBMSCs培养液中含有NGF,随培养时间的延长,培养液中NGF的浓度增加;hBMSCs后期诱导液中也含有NGF,且诱导后各时间点浓度均明显高于诱导前(P<0.01)。结论:hBMSCs在贴壁培养以及向神经细胞诱导分化过程中,均可向外分泌NGF等神经营养因子,且诱导后分泌量明显增加。

Experimental study on expression changes of NGF during the differentiation of human bone marrow stromal stem cells into nerve cells

Objective:To detect the expression of NGF in supernatant of inducing medium from hBMSCs to nerve cells,and explore the mechanism of BMSCs transplantation.Methods:hBMSCs harvested from adult normal human marrow,then were passaged and purified in culture medium.hBMSCs were passaged three times before experiment,then purified hBMSCs were induced by SHH(sonic hedgehog,400ng/L),RA(retubiuc acids,0.5 μmol) and Fn(forskolin,5 μmol).The concentration of NGF in supernatant was detected by ELISA on 1 d,3 d,5 d,7 d in Supernatant of hBMSCs culture medium,then we analyzed and compared the concentrations of NGF between before and after induction at three time points(12 h,1 d,3 d).Results:NGF was tested in supernatant of hBMSCs culture medium.Along with time prolonging,the concentration of this neurotrophic factor increased.We had also found NGF in supernatant of inducing medium,moreover,the concentration at every time point after induction was higher than before(P<0.01).Conclusion:hBMSCs can secrete NGF both in adherent culture and inducing culture,furthermore,the secretion increased significantly in latter culture.