不同时间诱导后上清液在骨髓间充质细胞向神经元样细胞分化中的作用

Effect of Supernatant after Induction by Sonic Hedgehog Retinoic Acids and Forskolin Inducible System on Human Bone Marrow Stromal Cells

目的研究体外使用音猬因子(SHH)、维甲酸(Ra)、Forskolin(Fn)组成的诱导体系诱导骨髓间充质细胞(BMSCs)后的上清液对BMSCs向神经细胞分化的作用。方法从健康志愿者髂骨处抽取骨髓4mL,体外分离、培养、扩增后用400ng/LSHH、0.5μmRa和5μmFn组成的诱导体系进行诱导,取诱导后6h、12h、24h、3d、5d和7d的诱导液上清分别作用于BMSCs。7d后使用倒置显微镜和免疫组织化学方法观察诱导前后细胞的变化。结果诱导7d后各组均有部分细胞胞体收缩、伸长突起,表现出神经细胞的形态。12h上清诱导组同SHH+Ra+Fn诱导组NSE、MAP-2阳性细胞比例高于其他组(P<0.05),12h上清诱导组同SHH+Ra+Fn诱导组NSE、MAP-2阳性细胞比例无统计学意义(P>0.05)。结论12h后上清液可有效诱导BMSCs向神经细胞分化。

Objective To investigate the effect of supernatant after induction by sonic hedgehog, retinoic acids, forskolin inducible system on differentiation of human bone marrow stromal cells （BMSCs）. Methods BMSCs from adult normal human marrow were purified in culture medium. Purified BMSCs were induced by sonic hedgehog （SHH）, retinoic acids （Ra） and Forskolin （Fn）. Supematant after induction were harvested on 6 h, 12 h, 24 h, 3 d, 5 d and 7 d, then purified BMSCs were induced by supematant after induction of 6 h, 12 h, 24 h,3 d,5 d and 7 d. BMSCs were detected with immunoeytochemisty staining for micmtubule associated protein - 2 （MAP - 2） and neuron specific enolase （NSE） 7 days later. Results After 7 days of induction, some BMSCs exhibited immunoreactivity. MAP -2 and NSE positive cells existed in all groups, while NSE, MAP- 2 and NSE positive cells increased markedly in BMSCs treated with supematant after induction of 12 h（P〈0.05）. Conclusion Supernatant after induction of 12 h could induce effectively human BMSCs to differentiate into neural cells in vitro.