摘要
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 study the role of Wnt / β-catenin signaling pathway in neural differentiation of rat BMSCs, Explore possible signal molecular mechanism in application of EGF and bFGF induce BMSCs to neuronal differentiation.Methods BMSCs were harvested by the whole bone marrow adherence method, BMSCs at passage 3 were respectively treated with 20ng/ml basic fibroblast growth factor （bFGF） and 20ng/ml epidermal growth factor （EGF） in containing 10ml / L （1%） fetal bovine serum （FBS） DMDM/F-12 medium. Then the cellular morphology was observed under inverted phase contrast microscope.At 7days after induction, Immunohistochemical staining was used to detect neuron specific enolase（NSE） and collagen fibre acidic protein（GFAP）,and RT-PCR to detect expression of β-catenin、BDNF、GDNF、nestin genes.Results At 7days after induction, BMSCs in group bFGF and EGF＋bFGF become round, extend multiple distinct processes around and have connections among some processes,but group EGF and control did not change significantly. Immunohistochemical staining show GFAP positive rate higher than NSE in group EGF,but in group bFGF and EGF＋bFGF NSE positive rate higher than GFAP. NSE and GFAP positive rate is highest in group EGF＋bFGF, group bFGF is followed, compare With blank group and group EGF,the differences were statistically significant （P〈0.0042）.RT-PCR show mRNA expression of nestin in group bFGF and EGF＋bFGF were significantly higher than other groups（P〈0.05）; β-catenin、BDNF in group EGF、bFGF and EGF＋bFGF were significantly higher than blank group and differences among these three groups were significant （P〈0.05）. GDNF in group bFGF and EGF＋bFGF were significantly higher than other groups and group bFGF was significantly higher than group EGF＋bFGF （P〈0.05）. Conclusion bFGF and EGF are able to induce differentiation of BMSCs into neural cells, the genes of β-catenin、BDNF and GDNF increase expression during differentiation. Wnt / β-catenin signaling pathway ma
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2013年第3期61-67,共7页
China Biotechnology
基金
江西省科技支撑计划项目(20111BBG70022-2)
