皮肤源祖细胞体外诱导神经元样细胞的实验研究

Differentiation of skin-derived precursors into neuron-like cells in vitro

目的探讨皮肤源祖细胞的体外培养、鉴定及定向诱导成神经元的方法,为治疗中枢神经系统疾病提供理想的种子细胞。方法取出生1～3d的昆明鼠皮肤,分离培养出皮肤源祖细胞;皮肤源祖细胞以含碱性成纤维细胞生长因子(bFGF)和10%胎牛血清(FBS)的培养基中预诱导24h,再以含二甲基亚砜(DMSO)和3-叔丁基-4-羟基茴香醚(BHA)的培养基中诱导7d;观察诱导细胞形态,以及免疫荧光鉴定细胞表达神经丝蛋白(NF)、神经元特异性烯醇化酶(NSE)和胶质原纤维酸性蛋白(GFAP)情况;ELISA检测诱导细胞分泌神经生长因子(NGF)和脑源性神经营养因子(BDNF)情况。结果细胞诱导后呈多极性生长;细胞免疫荧光表达NF和NSE,GFAP表达阴性;随诱导时间延长培养液中BDNF和NGF浓度增加。结论皮肤源祖细胞在特定条件下能定向分化为神经元,并且能提高神经生长因子和脑源性神经营养因子的分泌,能为组织工程皮肤和修复神经系统病变提供种子细胞。

Objective To explore methods to culture and isolate skin-derived precursors （SKPs）and direct them to differentiate neuron-like cells, so as to provide ideal seeded cells for central nervous system disease treatment. Methods Skin from juvenile Kunming mice was dissociated and cultured for skinderived precursors. Those precursors were pre-induced 24 hours in medium contained with basic fibroblast growth factor （bFGF） and 10% fetal bovine serum. Then cells were differentiated with dimethyl sulphoxide （DMSO） and butylated hydroxyanisole （BHA）. Growth status of SKPs was observed and immunofluorescent techniques were used to detect neurofilament protein （NF）, neurone specific enolase （NSE） and glial fibrillary acidic protein （GFAP） marker expression. Then nerve growth factor （NGF） and brain-derived neurotrophic factor （BDNF） were tested by enzyme-linked immunospecific assay to detect cells secretion. Results After differentiation for neuron-like cells, cells were transformed to multipolar. NF and NSE marker expression was deteced, but GFAP was not. The concentration of NGF and BDNF was significantly higher compared with the controls. Conclutions SKPs can be induced to differetiate into neuron-like cells in vitro. NGF and BDGF would be secreted after differentiation. The ability of skps to isolate and expand such a stem cell from an accessible, potentially autologous tissue source has important therapeutic implicationsfor tissue engineering.