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目的观察两种方法体外诱导大鼠骨髓间充质干细胞(BMSC)向神经样细胞分化的效果。方法自大鼠股骨中提取骨髓细胞,利用Percoll法进行BMSC分离、纯化及鉴定。取第5代细胞,应用两种方法进行诱导。方法一(BHA组):200μmol/L丁羟基茴香醚(BHA)、10 ng/ml碱性成纤维生长因子(bFGF)和2%二甲基亚砜(DMSO)联合诱导细胞;方法二(RA组):全反式维甲酸(RA)和β-巯基乙醇(β-ME)联合诱导细胞。同时设正常培养的BMSC为对照组。实验过程中对细胞进行形态观察和免疫荧光染色检测神经细胞标志物。结果BHA组诱导5 h,细胞即发生明显的形态改变,出现胞体回缩、突触伸展等神经干细胞的形态特征,而RA组诱导1周未出现神经干细胞形态。免疫荧光染色显示两种方法诱导的细胞均能表达神经前体细胞标志物nestin、β-tubulin和成熟神经元标志物神经元特异性烯醇化酶(NSE),不表达星形胶质细胞标志物胶质原纤维酸性蛋白(GFAP)。BHA组与RA组表达NSE的阳性率分别为80.05%和71.61%,两者相比,差异无统计学意义(P>0.05);对照组不表达NSE,诱导组与对照组比,差异有统计学意义(P<0.05)。结论两种方法均能诱导骨髓间充质干细胞定向分化为表达神经细胞标志物的神经样细胞。
Objective To observe the effects of two methods on differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells in vitro. Methods Bone marrow cells were extracted from the femur of rats. The BMSCs were isolated, purified and identified by Percoll method. Take the 5th generation of cells, the use of two methods for induction. Method one (BHA group): 200μmol / L butylated hydroxyanisole (BHA), 10ng / ml basic fibroblast growth factor (bFGF) and 2% dimethyl sulfoxide (DMSO) Group): All-trans retinoic acid (RA) and β-mercaptoethanol (β-ME) cells were induced in combination. At the same time set the normal culture of BMSC as control group. During the experiment, cells were observed morphologically and immunofluorescently to detect neuronal cell markers. Results The morphology of neural stem cells was observed in BHA group after 5 h of induction. Morphological characteristics of neural stem cells such as cell body retraction and synaptic extension were observed. However, the morphological changes of neural stem cells were not found in RA group. Immunofluorescence staining showed that the cells induced by both methods could express neural precursor cell markers nestin, β-tubulin and mature neuron marker neuron-specific enolase (NSE) without expressing astrocyte markers Glial fibrillary acidic protein (GFAP). The positive rates of NSE expression in BHA group and RA group were 80.05% and 71.61% respectively, there was no significant difference between the two groups (P> 0.05); NSE was not expressed in the control group, and there was statistical difference between the induction group and the control group Significance (P <0.05). Conclusion Both methods can induce bone marrow mesenchymal stem cells to differentiate into neuron-like cells that express neural cell markers.