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目的:研究Wnt/β-catenin信号通路在大鼠BMSCs神经分化中的作用,探讨应用EGF、bFGF诱导BMSCs神经分化的可能信号分子机制。方法采用全骨髓贴壁法体外分离纯化大鼠BMSCs。第3代BMSCs分别给予20ng/ml碱性成纤维生长因子(bFGF)和(或)20ng/ml表皮生长因子(EGF)在含10ml/L(1%)胎牛血清(FBS)的DMDM/F-12培养基中诱导,倒置相差显微镜观察其形态变化,免疫组织化学法检测细胞内神经元特异性烯醇化酶(NSE)及胶质纤维酸性蛋白(GFAP)的表达,RT-PCR检测其β-catenin、BDNF、GDNF、nestin基因mRNA的变化。结果诱导7d后bFGF和EGF+bFGF组细胞变圆,向四周伸出多个明显突起,部分突起间存在连接,而EGF组、空白组变化不明显;免疫组化染色示EGF组GFAP阳性率高于NSE,而bFGF和EGF+bFGF组NSE阳性率高于GFAP。EGF+bFGF组NSE、GFAP阳性率最高,bFGF组次之,与空白组、EGF组比较差异均有统计学意义(P<0.004 2);RT-PCR示nestin在bFGF和EGF+bFGF组较其他组显著增高,差异均有统计学意义(P<0.05);β-catenin、BDNF在EGF组、bFGF组、EGF+bFGF组显著高于空白组,且各组间比较差异均有统计学意义(P<0.05);GDNF在bFGF组、EGF+bFGF组显著高于其他组,且bFGF组高于EGF+bFGF组,差异均有统计学意义(P<0.05)。结论 bFGF、EGF可诱导大鼠BMSCs向神经细胞分化,分化过程中β-catenin、BDNF、GDNF基因表达增高,Wnt/β-catenin信号通路可能在BMSCs向神经分化过程中起重要作用。
OBJECTIVE: To investigate the role of Wnt / β-catenin signaling in the neural differentiation of BMSCs in rats and to explore the possible molecular mechanism of the induction of neural differentiation of BMSCs using EGF and bFGF. Methods BMSCs were isolated and purified by whole bone marrow adherent method in vitro. The third generation of BMSCs were treated with 20 ng / ml basic fibroblast growth factor (bFGF) and / or 20 ng / ml epidermal growth factor (EGF) in DMDM / F with 10 ml / L fetal bovine serum -12 medium, the morphological changes were observed under inverted phase contrast microscope, the expression of intracellular neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) was detected by immunohistochemistry, and the expression of β -catenin, BDNF, GDNF, nestin mRNA. Results After induction for 7 days, the cells in bFGF and EGF + bFGF groups became round, with many prominent protrusions extending around them, and some protrusions were connected with each other. However, EGF and blank groups showed no obvious changes. Immunohistochemical staining showed that the positive rate of GFAP in EGF group was high In NSE, while the positive rate of NSE in bFGF and EGF + bFGF group was higher than that in GFAP. The positive rates of NSE and GFAP in EGF + bFGF group were the highest, followed by bFGF group, and there was significant difference compared with blank group and EGF group (P <0.004 2); RT-PCR showed nestin was higher in bFGF and EGF + bFGF group than other groups (P <0.05). The expressions of β-catenin and BDNF in EGF group, bFGF group and EGF + bFGF group were significantly higher than those in blank group (P <0.05) P <0.05). The levels of GDNF in bFGF group and EGF + bFGF group were significantly higher than those in other groups, and the difference was statistically significant (P <0.05). Conclusion bFGF and EGF can induce the differentiation of rat BMSCs into neurons. The expression of β-catenin, BDNF and GDNF is increased during the differentiation. The Wnt / β-catenin signaling pathway may play an important role in the differentiation of BMSCs into neurons.