人Six1基因促进肺腺癌细胞A549的增殖  

Human Six1 Gene Promotes the Proliferation of Lung Cancer Cell A549 Lines

作　　者：陈思禹[1] 李玲[2] 晋帅 洪甜[2] 黄俊[3] 尤文叶 韩笑 崔越[5] 叶棋浓[2] 王钰琦[1] 

机构地区：[1]解放军总医院,北京100853 [2]军事医学科学院生物工程研究所,北京100850 [3]军事医学科学院附属医院,北京100071 [4]锦州医科大学,辽宁锦州121000 [5]首都师范大学,北京100048

出　　处：《生物技术通讯》2017年第2期96-100,127,共6页Letters in Biotechnology

基　　金：国家自然科学基金(81472589)

摘　　要：目的:构建带有Flag标签的Six1基因的慢病毒表达载体,并对表达产物Flag-Six1的生物学功能进行初步鉴定。方法:以实验室构建的真核表达载体myc-Six1为模板,根据实验需求合成引物,经PCR扩增,酶切后插入pCDHFlag载体,Western印迹检测其在人293T细胞中的表达;经Western印迹鉴定无误后,与3个包装质粒共转染293T细胞,包装成慢病毒后感染人肺癌细胞A549,经嘌呤霉素筛选2周,收集部分细胞,用Western印迹验证蛋白水平的表达,并通过细胞生长实验检测Six1对A549细胞生长的影响;提取RNA,通过qRT-PCR检测细胞中血管内皮细胞生长因子C(VEGF-C)在mRNA水平的变化。结果:构建的慢病毒表达载体pCDH-Flag-Six1能够在293T细胞中正确表达;包装成慢病毒,感染肺癌细胞A549后可正常表达;生长曲线表明Flag-Six1可促进人肺癌细胞A549的繁殖;qRTPCR结果表明Six1可升高肺癌细胞A549的VEGF-C转录水平。结论:构建了带pCDH-Flag标签的人Six1基因慢病毒表达载体,Flag-Six1能够在肺癌细胞系A549中表达,并能促进细胞增殖,这为进一步研究Six1的生物学功能奠定了重要基础。Objective： To construct the lentiviral vector of human Sixl labeled with CDH-Flag and preliminary detect the biological function of the expression product CDH-Flag-Sixl. Methods： Human Sixl gene was obtained from myc-Sixl plasmid by PCR and cloned into pCDH-Flag vector. In the human kidney 293T cells, the expression of the constructed pCDH-Flag-Sixl was identified by Western blotting. The recombinant plasmid pCDH-Flag- Sixl was co-transfected with three package plasmids into 293T ceils, and then the supernatant containing lentivirus infected lung cancer A549 cell lines. After two weeks of puromycin screening, stable A549 cell lines were hatvested and subjected to Western blotting, growth curve experiment and qRT-PCR to identify Sixl expression, examine growth condition, and mRNA level of VEGF-C respectively. Results： The growth curve showed Sixl overexpressing A549 cell lines accelerated proliferation. The qRT-PCR showed human Sixl gene could up-regulate the VEGF-C mRNA expression in lung cancer A549 cells. Conclusion： The lentiviral vector of pCDH-Flag-Sixl has been constructed and the CDH-FXag-Sixl protein promotes the proliferation and regulates VEGF-C expression, which has laid foundation for the further study on the function of Sixl.

关 键 词：人Six1基因 慢病毒表达载体 生物学功能 

分 类 号：Q78[生物学—分子生物学] Q25