人EGFL7基因RNA干扰慢病毒载体的构建及其在人喉癌细胞中的表达  

Construction of a lentiviral vector for RNA interference of human EGFL7gene and its stable expression in human laryngeal carcinoma cells

作　　者：张革化[1] 常利红[1] 叶进[1] 庄士民[1] 王凯[1] 黎景佳[1] 

机构地区：[1]中山大学附属第三医院耳鼻咽喉头颈外科,广东广州510630

出　　处：《中国病理生理杂志》2013年第10期1864-1869,共6页Chinese Journal of Pathophysiology

基　　金：高校基本科研业务费中山大学青年教师培育计划(No.11ykpy43);广东省自然科学基金博士启动基金资助项目(No.S2012040006622);广东省对外合作项目(No.2012B050600015)

摘　　要：目的:构建人表皮生长因子样结构域7(epidermal growth factor-like domain 7,EGFL7)基因RNA干扰(RNA interference,RNAi)慢病毒表达载体,并建立稳定干扰EGFL7基因表达的喉癌细胞亚系,为研究EGFL7蛋白在喉癌发生发展中的功能奠定基础。方法:针对人EGFL7基因序列,设计特异性RNAi靶序列,与pcDNA6.2-GW/EmGFP-miR线性载体定向连接,得到的阳性重组子再与pLenti6.3-MCS/V5-DEST慢病毒载体进行重组,获得真核表达慢病毒干扰载体。在POLOdelivererTM3000介导下将慢病毒包装质粒和EGFL7基因重组慢病毒载体导入293T细胞包装病毒,测定病毒滴度,感染人喉癌细胞HEp-2,杀稻瘟菌素筛选获得稳定干扰EGFL7基因的细胞亚系。结果:成功构建EGFL7 pLenti6.3-EGFL7-miR真核表达慢病毒干扰载体并获得相应慢病毒,经测定病毒滴度为5×1011TU/L,实时荧光定量PCR证实pLenti6.3-EGFL7-miR慢病毒载体对喉癌细胞HEp-2 EGFL7基因的沉默效率为97%。结论:成功构建人EGFL7基因特异性的慢病毒干扰载体,并获得EGFL7基因稳定干扰的喉癌细胞亚系。AIM：To investigate the role of epidermal growth factor-like domain 7 （EGFL7） in the pathogenesis and progress of laryngeal carcinoma via constructing a lentiviral expression vector for RNA interference （RNAi） of human EGFL7 gene and assessing the gene-silencing effect of the vector in human laryngeal epidermoid carcinoma （HEp-2） cells. METHODS：Specific RNAi target sequences were designed focused on human EGFL7 gene sequence. The double-stranded oligonucleotides were cloned into the pcDNA6.2-GW/EmGFP-miR plasmid after synthesis and annealing. A positive clone was subcloned into the pLenti6.3-MCS/V5-DEST vector after sequence analysis. The recombinant lentivirus was harvested from 293T cells co-transfected with the positive recombinant plasmid and lentiviral packing materials. HEp-2 cells were infected with the recombinant lentivirus and the cells with stable EGFL7 knockdown were screened by blasticidin selection. EGFL7 mRNA expression in the cells was determined by real-time fluorescence quantitative PCR. RESULTS：A recombinant lentiviral vector expressing short hairpin RNAs （shRNAs） against EGFL7 gene was obtained and confirmed by DNA sequencing. The virus titer was 5×1011 TU/L, and the silencing efficiency was 97%. CONCLUSION：A lentiviral vector targeting human EGFL7 gene, capable of stable EGFL7 gene knockdown in HEp-2 cells, has been successfully constructed, which provides a basis for further study of the relationship between human laryngeal carcinoma and EGFL7 protein.

关 键 词：EGFL7蛋白 RNA干扰 喉肿瘤 慢病毒 

分 类 号：R363[医药卫生—病理学]