人骨髓基质细胞分化为神经细胞潜力的研究

The study of potential of human bone marrow stromal cell(hMSCs) differentiating into neural cell in medium with serum

目的 研究骨髓基质细胞在含血清培养基中向神经细胞分化的潜能。方法 从健康人肋骨分离原代骨髓基质细胞 ,接种于含 10 %胎牛血清 (FBS)的DMEM中 ,分为A组 (3～ 4d换液一次 )、B组 (不换液 ) ,在相差显微镜下观察生长情况。在培养的 1,5 ,10d用免疫荧光法检测两组骨髓基质细胞的各种表面标志物巢蛋白 (nestin)、胶质纤维酸性蛋白 (GFAP)、微管相关蛋白 (MAP 2 )、S 10 0表达情况。并在培养的第 1,4 ,7,10 ,13d用酶联免疫吸附法 (ELISA)检测B组培养细胞上清中的脑源性神经营养因子 (BDNF)和神经生长因子 (NGF)的分泌水平。结果 A组细胞分裂繁殖速度明显高于B组 ,在培养的第 11～ 12d ,B组中部分细胞表现为胞体锥形并有长突起的神经元样形态 ,且形成网状。A组中随着培养时间的延长仅仅有nestin表达增加 ,B组中Nestin ,GFAP ,MAP 2的表达随时间的不同有差异 ,S 10 0在体积较小的细胞表达较强。B组细胞培养的第 10d ,检测到BDNF的表达 ,培养第 7,10 ,13d检测到NGF分泌。结论 人骨髓基质细胞在含血清培养基中能够表达神经前体细胞和神经细胞的相关表面标志物 ,并且能够分泌一定水平的神经营养因子 ,表现出可分化成神经细胞的潜能。血清和骨髓基质细胞自身分泌物质的蓄积可能在其中起到了重要的作用。

Objective To study the types and ability of human bone marrow stromal cell(hMSCs) differentiating into neural cell in medium with serum.Methods hMSCs from healthy person were propagated in DMEM with 10% fetal bovine serum(FBS).Medium was renewed every three days in group A, but no change in group B.The growth was observed under contrast phase microscope. The epitope of hMSCs was detected with immunofluorescence at the culturing day 1,5,10. Brain derived neurotrophic factor (BDNF) and nerve growth factor(NGF) levels of group B were analyzed by enzyme linked immunoadsordent assay(ELISA) at the culturing day 1,4,7,10,13.Results The growth of group A is faster than that of group B. At the 11th day, a few cells of group B displayed cone shape body and long processes as neuron. The expression of nestin in group A was found in different culturing time. The different expressions of nestin, microtube associated protein 2(MAP 2) and glial fibrillary acidic protein(GFAP) in group B were observed in different culturing time.The expression of S 100 was more significant in smaller cells.In group B, BDNF was detected at the culturing day 10 and NGF was detected at the culturing day 7,10,13.Conclusion hMSCs may express the epitopes of neural cell and neural progenitor cell and secrete BDNF and NGF in the 10%FBS DMEM.hMSCs have the great potential to differentate into neural like cells in vitro. Serum and substance secreted by hMSCs may play an important role.