大鼠骨髓间充质干细胞体外分化为视网膜神经节样细胞的实验研究

Rat bone marrow stromal cells differentiation into retinal ganglion cell phenotype in vitro

目的探讨大鼠骨髓间充质干细胞(BMSCs)体外向视网膜神经节细胞(RGCs)方向分化的可能性。方法取成年大鼠BMSCs原代培养,传代后获得高纯度BMSCs。采用碱性成纤维细胞生长因子(bFGF)诱导法,相差显微镜和荧光黄(LY)细胞内染色观察诱导后细胞形态的变化,并采用免疫细胞化学法检测微丝微管相关蛋白-2(MAP-2)、Thy1.1和胶质纤维酸性蛋白(GFAP)的表达。结果诱导后BMSCs呈现神经元样外观。LY染色可见细胞的多级突起,部分邻近细胞出现LY染色阳性。诱导后的神经元样细胞MAP-2和Thy1.1表达阳性,GFAP表达阴性。结论大鼠BMSCs经bFGF体外诱导可分化为具有RGCs表型的神经元样细胞,诱导后细胞间存在突触联系。

Objective Bone marrow stromal cells （BMSCs） are muhipotent adult stem cells,and they offer a distinct advantage by having autologous immunological characteristics as well as a relatively easy isolation and expansion. Previous studies have shown that BMSCs can differentiate into osteoblasts, adipocytes, cardiac muscle cells and neural lineages. Our study was to investigate the differentiating potential of BMSCs into retinal ganglion cells （RGCs） in vitro. Methods BMSCs were obtained from femurs of rats （n = 10） and purified after 3 passages. The cells were then induced by cytokine （basic fibroblast growth factor ,bFGF） protocol （ 10 ng/mL） ,and growth medium （ DMEM/F12 ＋ 10% FBS） was used as a control. Morphological changes of inducted cells were monitored and observed by lucifer yellow intracellular injection. Immunocytochemistry was used to detect the expression of microtuble-associated protein （MAP-2） , Thy1. 1 and glial fibrillary acidic protein （GFAP）. Results The inducted cells exhibited neuronal-like shape and extended long processes. After lucifer yellow injection, the inducted cells formed dendritic processes,and the adjacent cells were also positively stained. These cells showed positive reaction for MAP-2 and Thy 1. 1 but absent reaction for GFAP. No obvious morphological change was seen and showed the absent staining for all cell markers in control cells. Conclusion BMSCs can differentiate into neuron-like cells with retinal ganglion cell phenotype after bFGF induction. Synapse may exist among the induced cells.