BMP-7诱导大鼠骨髓间充质干细胞向神经元细胞分化的初步研究

摘要

Objective To investigate the effect of bone morphogenetic protein 7 (BMP-7 ) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into neuronal cells in vitro. Methods BMSCs were isolated and cultured by whole bone marrow adherence method; Flow cytometry was used to detect the cell phenotype CD29, CD44 and adipogenic induction to identify the rat BMSCs; the effects of 25, 50, 75, 100 ng/ml of BMP-7 on the proliferation of rat BMSCs were detected by MTT assay; the third generation cells were randomly divided into three groups: negative control group, positive control group (β-mercaptoethanol), BMP-7 induction group; The morphological changes of the cells were observed under inverted phase contrast microscope; After induction of 2 h, the expression of neurofilament protein 200 (NF-200) and synaptophysin 1 (SYN-1) was detected by immunocytochemistry. Results The morphology of rat BMSCs in the 3rd generation was in the form of long spindle, "Crow's feet" shaped to grow;The positive rates of CD29 and CD44 were 99. 44％ and 99. 17％, respectively;Rat BMSCs oil red O staining was positive after 28 days of adipogenic induction; Different concentrations of BMP-7 had the effect of promoting the proliferation of rat BMSCs, and the pharmacological effects of the 75 ng/ml group were the most significant;Typical neuronal cells could be found after rat BMSCs were induced by 75 ng/ml BMP-7 for 2 h, NF-200 and SYN-1 was positive. Conclusion BMP-7 has the effect of inducing BMSCs to differentiate into neuronal cells in vitro.%目的 探讨骨形态发生蛋白7(BMP-7)体外诱导大鼠骨髓间充质干细胞(BMSCs)向神经元细胞分化的作用.方法 以全骨髓贴壁法分离、培养大鼠BMSCs;流式细胞仪检测细胞表型CD29、CD44及成脂诱导以鉴定大鼠BMSCs; MTT法检测浓度分别为25、50、75、100 ng/ml的BMP-7对大鼠BMSCs增殖的影响;第3代细胞贴壁后随机分为三组:阴性对照组、阳性对照组(β-巯基乙醇)、BMP-7诱导组;倒置相差显微镜下观察各组细胞形态学变化;诱导2 h后进行免疫细胞化学实验,检测各组细胞神经元标志物神经丝蛋白-200(NF-200)及神经突触素-1(SYN-1)的表达情况.结果 体外培养的第3代大鼠BMSCs形态呈长梭形,"鱼尾纹"状聚集生长;CD29、CD44阳性表达,阳性率分别为99.44％、 99. 17％;成脂诱导28 d后油红0染色阳性;不同浓度的 BMP-7均具有促进大鼠BMSCs增殖的作用,以75 ng/ml最为显著;75 ng/ml BMP-7诱导大鼠BMSCs 2 h后可见形态典型的神经元细胞,NF-200及SYN-1为阳性.结论 BMP-7体外具有诱导大鼠BMSCs向神经元细胞分化的作用.