沉默核仁素可诱导大鼠神经干细胞的分化

摘要

目的 探讨核仁素(nucleolin)对原代神经干细胞(NSCs)分化的影响及其相关机制.方法 构建核仁素siRNA腺病毒表达载体,将经鉴定正确的重组腺病毒质粒转染HEK293A细胞,包装得到具有感染能力的nucleolin-siRNA重组腺病毒.病毒体外感染原代NSCs,采用Western blot检测nucleolin蛋白的表达情况,Nucleolin-siRNA对神经干细胞Nestin、Wnt3、β-catenin蛋白表达的影响;采用免疫细胞化学检测nucleolin-siRNA对神经干细胞分化的影响.结果 经酶切和测序鉴定均证实nucleolin-siRNA重组腺病毒载体构建成功,其插入序列正确无误.Western blot检测结果显示,转染nucleolin-siRNA2的细胞nucleolin表达明显受到抑制.siRNA+组NSCs分化为NSE阳性细胞和GFAP阳性细胞的分化率明显比CON组、siRNA-组增加,且差异具有统计学意义(P<0.01).与阴性对照组相比,siRNA+组Nestin的表达明显减少,Wnt3表达明显增多,β-catenin入核明显增多,其差异具有统计学意义(P<0.05).结论 本研究成功地构建了针对nucleolin基因的siRNA重组腺病毒载体,沉默nucleolin可诱导神经干细胞的分化,其机制可能与Wnt信号转导通路的激活有关.%Objective To investigate the effect of nucleolin silencing on the differentiation of rat neural stem cells (NSCs) and the role of Wnt signaling pathway in mediating such effect. Methods Adenovirus vectors expressing small interfering RNA (siRNA) against nucleolin were constructed, verified, and packaged in HEK293A cells. The adenovirus was then transfected into NSCs isolated from neonatal SD rats and the differentiation of the NSCs was examined by detecting the expressions of neuron specific encloase (NSE) and glial fibrillary acidic protein (GFAP) using immunocytochemistry. The expressions of nucleolin, nestin, Wnt3, and β-catenin in the cells were determined with Western blotting. Results Restriction endonuclease and sequencing analysis verified successful construction of the adenoviral vector expressing nucleolin siRNA (nucleolin-siRNA2). Infection of rat NSCs with nucleolin-siRNA2 significantly lowered nucleolin protein expression as compared with that in negative and blank control groups (P<0.05). The percentages of NSE-positive cells and GFAP-positive cells were significantly higher in NSCs infected with nucleolin-siRNA (P<0.01);the infection also resulted in obviously lowered expression of nestin protein and increased expressions of Wnt3 protein andβ-catenin nucleoprotein in the cells. Conclusions Nucleolin silencing by adenovirus-mediated RNA interference induces the differentiation of NSCs into neurons and astrocytes, which is related with the activation of Wnt signaling pathway.