骨髓间充质干细胞分化为内耳毛细胞前体细胞的研究

Differentiation of bone marrow mesenchymal stem cells into inner ear hair cell progenitors in vitro

目的 探索大鼠骨髓间充质干细胞（MSCs）体外诱导分化成内耳毛细胞前体细胞的过程,以期为耳蜗MSCs移植提供实验依据.方法 采用贴壁法提取SD大鼠MSCs,体外培养纯化.分别以EGF＋IGF-1＋BDNF＋ATRA、EGF＋IGF-1＋bFGF＋BDNF＋NT-3作为诱导条件,诱导MSCs分化.观察诱导组及对照组细胞形态的变化,并采用免疫细胞染色法检测毛细胞前体细胞或毛细胞特异性抗原myosinVI、Pax-2、nestin、p27kip1的表达.结果 EGF＋IGF-1＋BDNF＋ATRA诱导组细胞呈短梭形样、方形样,未形成突触;myosin VI、Pax-2、nestin、p27kpi1均有不同程度阳性表达.EGF＋IGF-1＋bFGF＋BDNF＋NT-3诱导组细胞变圆或变细长,逐渐伸出突触,部分形成网络状、大环状结构;nestin、p27kpi1均呈阳性表达.结论 大鼠MSCs在体外培养诱导分化后,可形成内耳毛细胞前体细胞和神经前体细胞.

Objective To investigate the differentiation the bone marrow mesenchymal stem cells （MSCs） into the hair cells progenitors in vitro. Methods MSCs were collected from rat bone marrows by plastic adherent selection method. After culture and purification in vitro,the combinations of EGF＋IGF-I＋BDNF＋ATRA or EGF＋IGF-I＋bFGF＋BDNF＋NT-3 were applied as inducing factors. The morphology of induced cells was observed, and the differentiated cells were characterized by immunocytochemical staining for the hair cell progenitors or hair cells-specific antigens nestin, Pax-2, myosin VI and p27kpl. Results With the combination of EGF＋IGF-1＋BDNF＋ATRA,the induced cells showed short spindle-like or rectangle-like appearance,but no processes formed; the cells positively expressed with myosin VI, Pax-2, nestin and p27kp1. With the combination of EGF＋IGF-I＋bFGF＋BDNF＋NT-3, the induced cells showed round or slim shape with gradually protruding processes; and parts of them formed net- or ring-like structure. Cells with processes demonstrated positive expression of nestin and p27kp1. Conclusion Rat bone marrow MSCs have the potential to differentiate into inner ear hair cell progenitors and neural progenitor cells in vitro.