【摘要】 目的利用CRISPR/Cas9技术构建Atg7基因敲除的人脑微血管内皮细胞,为Atg7在脑血管的发育和参与血脑屏障形态建成的机制以及脑血管相关疾病的研究提供体外细胞模型。方法针对人Atg7第1外显子区域设计2对引导RNA序列(sgRNA),通过慢病毒转染人脑微血管内皮细胞,嘌呤霉素和杀稻瘟菌素筛选成功转染细胞。分别采用实时荧光定量聚合酶链反应(PCR)和Western blotting法检测敲除靶基因的mRNA和蛋白,显微镜下观察细胞形态变化。结果构建了靶向Atg7的CRISPR/Cas9质粒;通过实时荧光定量PCR、蛋白免疫印迹和细胞免疫荧光染色检测,结果表明单克隆筛选获得了Atg7稳定敲除的人脑微血管内皮细胞系;光镜下观察未发现Atg7敲除后细胞形态出现明显改变。结论利用CRISPR/Cas9技术成功构建了Atg7基因稳定敲除的人脑微血管内皮细胞,将有利于脑血管的发育和血脑屏障形态建成的机制以及脑血管相关疾病的研究。更多还原

【Abstract】 Objective To construct Atg7-gene-knockout human brain microvascular endothelial cells(HBMEC) by CRISPR/cas9 technology, so as to provide an in vitro cell model for the role of Atg7 in the development of cerebral vessels, the mechanism of Atg7 participating in the formation of blood-brain barrier and the study of cerebrovascular diseases.Methods Two pairs of guiding RNA sequences(sgRNA) were designed according to exon 1 of human Atg7. HBMEC was transfected with lentivirus; puromycin and Pyricularia oryzae were used to screen for cells successfully transfected. RT-qPCR and Western blotting were used to detect the mRNA and protein of the target gene, and the morphological changes were observed under the microscope.Results The CRISPR/cas9 plasmid targeting Atg7 was constructed. The results of RT-qPCR, Western blot and immunofluorescence showed that stable Atg7-knockout human brain microvascular endothelial cell line was obtained by monoclonal screening. No obvious morphological changes were observed under light microscope.Conclusion The successful construction of human brain microvascular endothelial cells with Atg7 gene knockout by CRISPR/cas9 technology is beneficial to the development of cerebral vessels, the mechanism of blood-brain barrier morphogenesis and the study of cerebrovascular diseases.更多还原