基于CRISPR/Cas9技术AEG-1基因敲除神经细胞系的构建及AEG-1基因敲除介导的神经细胞周期阻滞和细胞凋亡抑制

星形胶质细胞上调基因-1(AEG-1)是近年来研究较多的癌基因，但在神经系统疾病方面研究尚少。AEG-1与神经退行性疾病有关，然而其具体作用机制尚不明确。本研究通过设计靶向AEG-1 sgRNA序列并合成相应寡核苷酸，将其克隆到GV392质粒中，构建sgRNA/Cas9二合一表达载体，并进行慢病毒包装纯化。用慢病毒感染小鼠海马神经元HT22细胞，进行药物筛选和sgRNA活性鉴定，建立稳定的AEG-1基因敲除的细胞系；并进一步观察神经元HT22细胞的增殖与凋亡能力。结果显示，成功构建了3种靶向AEG-1基因的sgRNA/Cas9二合一表达载体。所设计的sgRNA的插入序列和开放阅读框架完全正确，成功建立了AEG-1基因敲除的稳转神经细胞系。进一步研究表明，AEG-1敲除后的神经HT22细胞与正常神经HT22细胞相比，细胞突起数目减少， 细胞周期阻滞，细胞凋亡率减少。以上结果为后续进一步研究AEG-1与神经系统疾病关系奠定了基础。

Construction of the AEG-1 Knockout Neural Cell Line Based on the CRISPR/Cas9 Technique and Inhibition of Cell Cycle and Cell Apoptosis Mediated by AEG-1 Knockout

Astrocyte elevated gene-1(AEG-1) is an oncogene that has been linked with neurological diseases, but the specific mechanism remains unclear. Herein we designed the sgRNA targeting AEG-1 sequences and synthesized the corresponding oligonucleotides. The sgRNA/Cas9 two-in-one expression vector was constructed and cloned into the GV392 plasmid. The constructed plasmid was packaged and purified, and transfected into the HT22 cells. The puromycin was used for cell selection to construct the stable AEG-1-knockout cell lines and test the activity of sgRNA. Further studies were performed to observe the effect of AEG-1 on the proliferation and apoptosis of the HT22 cells. AEG-1 knockout neurons displayed decreased synapsis, arrested cell cycle, reduced cell proliferation, and decreased apoptosis compared with the normal neurons. These results implicate AEG-1 in the neurological diseases and lay a foundation for further research.