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本实验观察了骨髓基质细胞(BMSCs)经化学诱导向神经细胞分化的过程,并初步探索其分化机制。首先分离培养扩增纯化SD大鼠BMSCs,应用β-巯基乙醇(BME)对第4代BMSCs进行诱导分化,分化稳定后撤除诱导液继续常规培养2周。结果显示:诱导分化前,细胞呈现梭形,平行排列生长。诱导分化后,多数BMSCs伸出突起,并发出初级和次级分支,相互交织成网状结构,成典型的神经元样细胞形态,分化过程中有部分细胞漂浮死亡。撤除诱导液常规培养2周后,分化的细胞逐渐缓慢恢复到原来成纤维细胞样长梭状形态。透射电镜观察到分化后细胞具有发育早期神经元的超微结构特点,免疫组织化学方法证实,神经干细胞标志蛋白(Nestin)在诱导分化后即开始表达,1h达到高峰,此时Nestin阳性细胞率为(29.35±1.45)%,之后随时间逐渐下降,5h时降至很低;神经元细胞标志蛋白-神经元特异性烯醇化酶(NSE)在诱导分化前不表达,在诱导分化后随时间表达逐渐增强,5h时阳性细胞率最高为(57.53±2.63)%;撤除诱导液常规培养2周时,Nestin、NSE表达均为阴性。星形胶质细胞标志蛋白-胶质纤维酸性蛋白(Glial fibrillary acidic protein GFAP)测定在不同组、不同时间点均为阴性结果。提示,BMSCs体外经BME化学诱导可向神经元样细胞分化,不能分化为胶质细胞;分化机制可能是先分化为神经干细胞,再转分化为神经元样细胞,这一化学分化过程是迅速、短暂、可逆的,且对细胞有损伤,因此,化学诱导后的BMSCs不适宜做细胞疗法的种子细胞。
In this study, we observed the process of bone marrow stromal cells (BMSCs) differentiated into neurons by chemical induction and explored the differentiation mechanism. Firstly, BMSCs were isolated, cultured and purified from BMSCs. BMSCs were induced to differentiate with BME. After differentiation, BMSCs were removed and induced to continue for 2 weeks. The results showed that: before induction of differentiation, cells showed fusiform, parallel growth. After induction of differentiation, most of the BMSCs protrude and protrude, and give rise to primary and secondary branches, interwoven into a network structure, into a typical neuron-like cell morphology, cell differentiation during the death of some cells. After removing the inducing fluid for 2 weeks, the differentiated cells slowly recovered to the original fibroblast-like spindle-like morphology. Transmission electron microscopy showed that the differentiated cells had the ultrastructural features of early developmental neurons. Immunohistochemistry confirmed that the expression of Nestin in the differentiated cells began to peak at 1h, and at this time, the rate of Nestin-positive cells was (29.35 ± 1.45)%, then decreased gradually with time and decreased to very low level at 5h. Neuronal cell marker protein-NSE was not expressed before induction of differentiation, (57.53 ± 2.63)% at 5h. Nestin and NSE expression were negative when the inducing fluid was removed by conventional culture for 2 weeks. Glial fibrillary acidic protein GFAP was negative in different groups at different time points. These results suggest that BMSCs can differentiate into neuron-like cells in vitro after being induced by BME, and can not differentiate into glial cells. The differentiation mechanism may be differentiation into neural stem cells and then into neuron-like cells. The chemical differentiation process is rapid, Transient, reversible, and cell damage, therefore, chemically induced BMSCs are not suitable for cell therapy of seed cells.