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目的:Olig1基因修饰的神经干细胞(NSCs)移植治疗中枢神经脱髓鞘疾病具有重要的应用前景。本研究目的是构建大鼠olig1真核表达载体,并观察其过表达对大鼠NSCs向少突胶质细胞分化的影响。方法:采用RT-PCR,以新生大鼠脊髓RNA为模板,扩增olig1基因,定向克隆到pEGFP-N3载体中;用电穿孔方法转染pEGFP-N3-olig1表达载体至NSCs中,然后用RT-PCR鉴定olig1的表达,免疫荧光染色鉴定NSCs向少突胶质细胞的分化情况。结果:成功构建了pEGFP-N3-olig1真核表达载体,olig1在重组质粒转染的NSCs中能够高效表达。重组质粒转染的NSCs在体外诱导分化后,能够较空质粒转染的NSCs产生更多的少突胶质细胞(P<0.01)。结论:Olig1过表达能够显著促进大鼠NSCs向少突胶质细胞方向分化。
OBJECTIVE: Olig1 gene-modified neural stem cells (NSCs) transplantation has important application prospect in the treatment of demyelinating diseases of the central nervous system. The purpose of this study was to construct the eukaryotic expression vector of rat olig1 and observe its effect on the differentiation of rat NSCs into oligodendrocytes. Methods: The olig1 gene was amplified by RT-PCR from neonatal rat spinal cord RNA and cloned into pEGFP-N3 vector. The vector pEGFP-N3-olig1 was transfected into NSCs by electroporation, The expression of olig1 was identified by PCR and the differentiation of NSCs into oligodendrocytes was identified by immunofluorescence staining. Results: The eukaryotic expression vector pEGFP-N3-olig1 was successfully constructed and olig1 was highly expressed in the transfected NSCs. The recombinant plasmid transfected NSCs could produce more oligodendrocytes than the empty plasmid transfected NSCs after induced in vitro (P <0.01). Conclusion: Overexpression of Olig1 can significantly promote the differentiation of rat NSCs into oligodendrocytes.