β-catenin基因对脊髓源性神经干细胞分化的作用

Roles of β-catenin gene in differentiation of rat spinal cord derived neural stem cells

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 determine the role of β-catenin gene in the differentiation of spinal cord derived neural stem cells. Methods After the spinal cord tissue was harvested from fetal rats at embryonic day 14, single cell cloning technique was used to separate and amplify neural stem cells （NSCs）, which were identified with immunofluorescence assay. Then the cells were divided into 3 groups, that is, separately cultured NSCs （blank group）, adenovirus vector AdGFP infected NSCs （GFP group）, and Adβ-catenin adenovirus vector infected NSCs （β-catenin group）. Cell infection efficiency was observed by fluorescence microscopy. The mRNA expression of neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP） was detected by RT-PCR, and the locations of neurons nucleoprotein （NeuN） and GFAP were observed by immunofluorescence assay. Western blotting was used to detect the protein expression of NeuN and GFAP. Results The cells derived from fetal rat spinal cord tissue had the ability of continuous differentiation and cloning, and started to form a small number of cell clusters. The cells were irregular in shape after mechanical separation. The majority of early separated cells were positive to Nestin antigen. The mRNA expression level of NSE was significantly higher in β-catenin group than blank group and GFP group （P〈0.05）, and that of GFAP was lower than the other 2 groups （P〈0.05）. Immunofluorescence assay showed that the expression of NeuN was significantly higher in β-catenin group than blank group and GFP group （P〈0.05）, and it was mainly located in the nuclei, but the expression of GFAP was lower （P〈0.05）. Western blot results showed that NeuN was highly expressed in β-catenin group than blank group and GFP group, but GFAP was lowly expressed in β-catenin group （P〈0.05）. Conclusion β-catenin promotes the differentiation of spinal cord derived NSCs into neuron.