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目的探讨脑源性神经营养因子(BDNF)体外诱导骨髓间充质干细胞(MSCs)分化成神经元样细胞的作用及其对诱导后的神经元样细胞保护作用,为治疗神经系统疾病提供更优良的细胞。方法体外培养MSCs至第5代后,分别用BDNF、β-巯基乙醇(β-ME)诱导MSCs,诱导后的第1、3和6小时计算两组神经元样细胞数,对各时间点两组神经元样细胞阳性率进行了比较;诱导后行神经细胞标记抗体的免疫细胞化学、RT-PCR及蛋白质印迹鉴定。结果两组诱导剂诱导后的细胞均呈神经元样细胞的形态;BDNF组诱导分化后细胞的存活时间远较β-ME组长;BDNF组在诱导后6h近于诱导高峰,而β-ME在诱导2h即达高峰,但随后神经元样细胞渐死亡。免疫细胞化学结果显示在两组诱导后细胞的巢蛋白(Nestin)、神经细胞特异性烯醇化酶(NSE)、微管相关蛋白-2(MAP-2)、神经丝蛋白(NF)表达均呈阳性,而胶质纤维酸性蛋白(GFAP)表达呈阴性;RT-PCR显示NSE、NFL,MAP-2的mRNA表达阳性,GFAP有较弱的表达;蛋白质印迹可见两组诱导剂诱导的细胞均表达神经元的特异性标记抗体NSE。结论BDNF能单独诱导MSCs分化形成神经元样细胞,而且分化后细胞的存活时间长,有望用于治疗脑缺血、神经变性等疾病。
Objective To investigate the effect of brain derived neurotrophic factor (BDNF) on differentiation of bone marrow mesenchymal stem cells (MSCs) into neuron-like cells in vitro and its protective effect on neuron-like cells after induced, so as to provide more excellent treatment for neurological diseases Cell. Methods MSCs were cultured in vitro for 5 passages. MSCs were induced by BDNF and β-mercaptoethanol (β-ME) respectively. The numbers of neuron-like cells were calculated at 1, 3 and 6 hours after induction. The positive rates of neuron-like cells were compared. Immunocytochemistry, RT-PCR and Western blotting were used to detect the labeled antibody of neural cells. Results The cells in both groups showed neuron-like morphology. The survival time of BDNF group was longer than that in β-ME group. The level of β-ME Peaked at 2 h after induction, but then neuronal-like cells gradually died. The result of immunocytochemistry showed that the expression of Nestin, NSE, MAP-2 and NF in the two groups were all higher Positive, and glial fibrillary acidic protein (GFAP) expression was negative; RT-PCR showed NSE, NFL, MAP-2 mRNA expression was positive, GFAP weaker expression; Specific neuronal marker antibody NSE. Conclusion BDNF can induce the differentiation of MSCs to form neuron-like cells alone, and the long survival time of the differentiated cells is expected to be used to treat cerebral ischemia and neurodegenerative diseases.