丹参注射液诱导间质干细胞分化为神经元样细胞

目的]丹参注射液体外定向诱导成人骨髓间质干细胞(MSC)分化为神经元样细胞.方法]采用Ficoll-Paque液离心分离成人MSC,体外扩增,流式细胞仪检测MSC表面抗原表达,分别采用含丹参注射液或硫代甘油等试剂的无血清达乐伯克改良必需基本培养基(DMEM)诱导MSC分化为神经元,免疫组化鉴定神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-M)、胶质纤维酸性蛋白(GFAP)、巢蛋白(nestin)的表达.结果]成人骨髓间质干细胞在体外扩增原代可获得0.5×106,15代可获得(2～3)×1012个细胞,细胞流式细胞仪检测结果显示CD29、CD44、CD90、CD105、CD166表达阳性,CD11a、CD14、CD34、CD38、CD45、CD80、CD86为阴性.加入丹参或硫代甘油诱导后,MSC胞体收缩,突起伸出;免疫组化显示诱导出的神经元样细胞NSE、NF-M、nestin表达阳性,GFAP阴性.结论]丹参可以在体外诱导成人骨髓间质干细胞分化为神经元样细胞.

Differentiaton of Human Mesenchymal Stem Cells into Neuron-like Cells with Danshen Injection

To investigate the differentiation of human mesenchymal stem cells(hMSC) into neuron like cells with Danshen injection (Salvia miltiorrhiza). hMSC were separated from rib marrow with Ficoll Paque reagent and expanded in culture medium. To detect the surface antigens,the labeled cells were analysed on a FACScan flow cytometer. hMSC were induced to differentiate into neurons with DMEM/Danshen injection or DMEM/monothioglycerol respectively. Neuron specific enolase(NSE), neurofilament(NF), Nestin, glial fibrillary acidic protein(GFAP) were detected by immunohistochemistry. hMSC were expanded as undifferentiated cells in culture for more than 15 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast like morphology. These expanded attached MSC were uniformly positive for CD29?CD44?CD90?CD105?CD166 and didn't express CD11a ?CD14?CD34?CD38?CD45?CD80?CD86. Simple methods with Danshen injection induced hMSC exhibit a neuronal phenotype, expressing NSE, NF M, Nestin at 5 hours. But the neuron like cells didn't express the glial astrocyte marker GFAP. Conclusion] hMSC can be induced to differentiate into neurons with Salvia miltiorrhiza.