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目的构建真核表达载体pEGFP-N1/hVEGF121,并转染神经干细胞(neural stem cells,NSCs),以探究人血管内皮生长因子(vascular endothelial growth factor,hVEGF121)基因导入NSCs的表达变化。方法分离培养胎鼠NSCs,并行nestin、GFAP免疫染色鉴定。采用PCR法,以hVEGF121质粒为模板,扩增hVEGF121全长编码序列,克隆入载体pMD18-T,随后又将其亚克隆入pEGFP-N1载体中,构建其真核表达载体pEGFP-N1/hVEGF121,并以Fugene 6介导转染,将hVEGF121导入NSCs。倒置显微镜检测转染后的NSCs生长变化及hVEGF121在其中的表达等情况。图像分析技术计算转染效率。RT-PCR法对hVEGF121转染的NSCs进行鉴定。结果成功分离培养到NSCs,并予以鉴定。构建了hVEGF121的真核表达载体pEGFP-N1/hVEGF121,成功地将hVEGF121转染NSCs,其转染效率约50%。被转NSCs生长良好。转染hVEGF121基因的NSCs被诱导分化后,神经球的轴突和轴丘表达神经元标志物NF-200。结论成功地将hVEGF121克隆到pEGFP-N1载体中,并实现了hVEGF121基因在NSCs的表达。
Objective To construct eukaryotic expression vector pEGFP-N1 / hVEGF121 and transfect neural stem cells (NSCs) to investigate the expression of hVEGF121 gene in NSCs. Methods Fetal NSCs were isolated and cultured and identified by nestin and GFAP immunostaining. HVEGF121 was cloned into vector pMD18-T by PCR using hVEGF121 as a template, and then subcloned into pEGFP-N1 vector to construct its eukaryotic expression vector pEGFP-N1 / hVEGF121. Transfection of hVEGF121 into NSCs was performed with Fugene 6. Inverted microscope was used to detect the growth of transfected NSCs and the expression of hVEGF121 in them. Image analysis techniques to calculate transfection efficiency. The hVEGF121 transfected NSCs were identified by RT-PCR. The results were successfully isolated and cultured to NSCs and identified. The eukaryotic expression vector pEGFP-N1 / hVEGF121 of hVEGF121 was constructed, and hVEGF121 was successfully transfected into NSCs. The transfection efficiency was about 50%. Transferred to NSCs grew well. NSCs transfected with hVEGF121 were induced to differentiate after neurosphere axons and axons expressed neuronal markers NF-200. Conclusion hVEGF121 was successfully cloned into pEGFP-N1 vector and hVEGF121 gene was expressed in NSCs.