成年大鼠骨髓基质干细胞诱导分化为神经元样细胞不同方法比较的研究

Comparative study of inducing differentiation of adult rat bone marrow stromal cells into neurons by various neurotrophic factors and antioxidant

目的 研究成年大鼠骨髓基质干细胞 (BMSCs)诱导分化为神经元样细胞不同的方法 ,寻找它向神经细胞分化的最佳条件。 方法 取纯度较高的BMSCs,通过不同的神经营养因子诱导法和抗氧化剂诱导法 ,进行抗巢蛋白 (nestin)、神经元特异烯醇化酶 (NSE)、神经胶质纤维酸性蛋白 (GFAP)、酪氨酸羟化酶 (TH)免疫细胞化学染色 ,观察相应的阳性细胞数。结果 诱导第 3天A组 (EGF :表皮生长因子 ,bFGF :碱性成纤维细胞生长因子 ,RA :全反式维甲酸 ) ,B组 (GDNF :胶质细胞系源性神经营养因子 ,BDNF :脑源性神经营养因子 ) ,C组 (EGF ,bFGF ,GDNF ,BDNF和RA)的Nestin阳性细胞数较多 ,其中以C组最多 ,而D组 (抗氧化剂 )Nestin阳性细胞数少于前三组。A ,B ,C组的NSE ,GFAP染色阳性细胞数较D组少 ,但D组有部分细胞发生死亡。诱导第 7天A ,B ,C组的NSE ,GFAP阳性细胞数较第 3天时明显增多 ,C组最多 ,B组其次 ,Nestin阳性细胞数比例较第 3天时明显减少。而D组的NSE ,GFAP阳性细胞数少于其第 3天时 ;C组诱导成神经细胞比例较高 ,阴性对照组和空白对照组极少或无阳性细胞。此外 ,神经营养因子诱导法生成神经样细胞的比例都多于胶质样细胞。 结论 抗氧化剂诱导法分化诱导快 ,而神经营养因子诱导法分化诱导效率高 。

Objective To investigate the conditions of inducing adult rat bone marrow stroma cells (BMSCs) differentiation into neurons in order to find the best way. Methods High purified BMSCs were selected and induced into neurons through the methods of various neurotrophic factors and antioxidant. The number of different immunoreactive cells was detected by nestin, NSE, GFAP and tyrosine hydroxylase (TH) immunocytochemistry. Results At the third day after the induction, more nestin positive cells were found in group A (EGF, bFGF and RA), B (GDNF and BDNF) and C (EGF, bFGF, GDNF, BDNF and RA), which was the most in group C, while less in group D (antioxidant). NSE and GFAP positive cells were more in group D than those in group A ,B and C, but the cell death was detected in group D. In group A, B and C, the number of NSE and GFAP-positive cells was much more at the 7th day than that at the 3rd day after induction. The number of nestin-positive cells was significantly decreased at the 7th day than that at the 3rd day after induction. In group D the number of NSE and GFAP-positive cells was less at the 7th day than that at the 3rd day after induction. The number of neurons induced was the most in group C. There were few or no positive cells in negative control and no induction groups. The proportion of neurons was higher than that of glial cells in all groups of neurotrophic factor induction. Conclusion The antioxidant can rapidly induce BMSCs into neurons, whereas neurotrophic factors can induce BMSCs into neurons with higher efficiency and good cell behavior. The various neurotrophic factors may take synergic actions in proliferation and differentiation of BMSCs.