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目的探讨β-catenin基因对脊髓源性神经干细胞(neural stem cells,NSCs)分化的作用。方法分离E14胎鼠脊髓组织,单细胞克隆技术对其分离细胞扩增培养,免疫荧光对细胞进行鉴定。实验分为3组:单独培养的NSCs(空白组)、感染空载Ad GFP腺病毒的NSCs(GFP组)、感染Adβ-catenin腺病毒的NSCs(β-catenin组)。荧光显微镜观察细胞感染效率,RT-PCR检测神经元特异烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)mRNA表达,免疫荧光检测神经元核蛋白(Neu N)、GFAP的表达及定位,Western blot检测Neu N、GFAP的表达。结果胎鼠脊髓组织分离的细胞具有连续分化及克隆能力,形成少量细胞团,机械分离后细胞呈不规则球形;早期分离的大部分细胞Nestin抗原呈阳性;NSE mRNA在β-catenin组显著高于空白组及GFP组,GFAP mRNA表达均低于其他两组(P<0.05);免疫荧光显示β-catenin组Neu N的表达水平显著高于空白组及GFP组(P<0.05),主要定位于细胞核中,GFAP的表达相对较低(P<0.05);Western blot检测显示β-catenin组的Neu N表达显著高于空白组及GFP组,而GFAP的表达低于其他两组(P<0.05)。结论β-catenin可促进脊髓源性NSCs向神经元分化。
Objective To investigate the effect of β-catenin on the differentiation of spinal cord-derived neural stem cells (NSCs). Methods E14 fetal rat spinal cord tissue was isolated, single cell clone technique was used to amplify and culture the isolated cells, and the cells were identified by immunofluorescence. The experiment was divided into 3 groups: NSCs cultured in vitro (blank group), NSCs infected with Ad GFP without GFP (GFP group) and NSCs infected with Ad-catenin (β-catenin). The cell infection efficiency was observed by fluorescence microscopy. The expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) mRNA was detected by RT-PCR. The expression and localization of neuron nuclear protein (Neu N) and GFAP were detected by immunofluorescence. Western blot detection of Neu N, GFAP expression. Results The cells isolated from fetal rat spinal cord showed continuous differentiation and cloning ability, formed a small number of cells, and the cells were irregularly spheroidized after mechanical separation. The majority of cells isolated early showed a positive expression of Nestin antigen. The expression of NSE mRNA in β-catenin group was significantly higher than that of The expression of Neu N in β-catenin group was significantly higher than that in blank group and GFP group (P <0.05), mainly located in The expression of GFAP in nucleus was relatively lower (P <0.05). Western blot showed that the expression of Neu N in β-catenin group was significantly higher than that in blank group and GFP group, while the expression of GFAP was lower than that in other two groups (P <0.05) . Conclusion β-catenin can promote the differentiation of spinal cord-derived NSCs into neurons.