β-巯基乙醇和脑匀浆上清对骨髓间充质干细胞的神经诱导作用

Effects of β-mercaptoethanol and brain extract supernatant on differentiation of bone marrow mesenchymal stem cells into neuron like cells

目的探讨β-巯基乙醇(BME)和大鼠脑匀浆上清对大鼠骨髓间充质干细胞(MSCs)分化为类神经元样细胞的作用。方法采用全骨髓培养法从SD大鼠骨髓中分离培养MSCs,经贴壁法体外扩增、传代和流式细胞仪鉴定。①以1mmol/L浓度BME的含血清DMEM培养基预诱导24 h,再以含5 mmol/L BME的无血清DMEM培养基诱导5 h至MSCs神经分化。②使用大鼠脑匀浆上清诱导MSCs,48 h至神经分化。免疫细胞化学染色分别检测两种诱导方法MSCs培养物神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)的表达。结果第5代MSCs有97.5%表达CD90抗原,1.72%表达CD45抗原。两种方法诱导后的细胞均具有神经细胞的形态特征;相比脑匀浆上清BME诱导组细胞形态变化更快。两种方法都可诱导MSCs表达NSE;鼠脑匀浆上清可诱导MSCs表达GFAP。结论MSCs具有向神经细胞分化的可塑性,相比BME,鼠脑匀浆上清具有诱导MSCs向胶质细胞分化的倾向。

Objective To investigate the effects of β-mercaptoethanol（BME） and rat brain extract supernatant on mesenchymal stem cells （MSCs） differentiation of rat into neuron like cells in vitro. Methods MSCs were obtained from SD rats and planted in plastic culture capsule, then cultured and expanded in vitro. MSCs were identified by flow cytometry. ① MSCs were maintained in serum-containing medium supplemented with 1 mmol/L BME for 24 h. And then, the cells were transferred to serum-free medium containing 5 mmol/L BME for 5 h to induce the differentiation into neuron like cells. ② MSCs were induced by incubation with rat brain extract supernatant in L-DMEM medium for 48 h to differentiate into neuron like cells. After induction, expression and differentiation rate of NSE, GFAP were detected with immunocytochemical method. Results 97.5% of the 5 passages of MSCs express CD90 and lack expression（ 1.72% positive） of CD45 ,The MSCs were induced into neuron like cells by two treatments,and the differentiated cells showed morphological characteristics of neuronal cells. Morphological modifications induced by BME were more dramatic compared with rat brain extract supernatant. In both treatments induced the expression of neural marker NSE. Rat brain extract supernatant induced the expression of GFAP. Conclusion MSCs can exhibit plastic neuro-differentiational potential in vitro. MSCs lean to differentiate into glia cells compared with BME.