CD147慢病毒表达载体的构建及稳定转染A549细胞系的建立  

Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

作　　者：杨绍兴[1] 汤传昊[1] 王思涵[1] 宋三泰 刘晓晴[1] 

机构地区：[1]军事医学科学院附属医院肺部肿瘤科,北京100071 [2]乳腺肿瘤科,北京100071

出　　处：《中国肺癌杂志》2012年第12期694-700,共7页Chinese Journal of Lung Cancer

基　　金：首都医学发展基金项目(No.2007-3042)资助~~

摘　　要：背景与目的 CD147是一类位于肿瘤细胞膜表面的跨膜糖蛋白,可促进肿瘤的浸润和转移。本研究拟构建CD147慢病毒表达载体,建立稳定过表达CD147的人肺腺癌A549细胞系,观察过表达CD147后对MMP-9及细胞增殖、侵袭能力的影响。方法 RT-PCR扩增CD147基因全长序列,将序列插入pEGFP载体,构建pEGFP-CD147慢病毒表达载体,随后转入293FT细胞中进行慢病毒包装,用获得的慢病毒毒液感染人肺腺癌细胞系A549,建立稳定过表达CD147的A549细胞系。Real-timePCR检测MMP-9的变化情况,CCK-8及Transwell法检测人肺腺癌细胞增殖、侵袭能力的变化。结果经限制性内切酶鉴定及测序分析,成功构建了pEGFP-CD147慢病毒表达载体质粒。Real-timePCR和Westernblot检测显示,与对照组相比,转染pEGFP-CD147慢病毒表达载体组的细胞,CD147的表达在mRNA和蛋白两个水平均增高,成功建立了A549-CD147细胞系。上调CD147的表达后,MMP-9的mRNA表达水平明显升高。同时,A549-CD147细胞增殖和侵袭能力明显增加(P<0.05)。结论成功构建CD147慢病毒表达载体和A549-CD147细胞系,过表达CD147可上调MMP-9的表达,增强人肺腺癌细胞的增殖和侵袭能力。Background and objective CD147, a type oftransmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD 147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction （RT-PCR）, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 ceils and establish a stable, overexpressed cell line named A549-CD 147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer ceils before and after transfection were examined by the CCK-8 and TransweU methods. Results A CD147 lentiviral expression vector （pEGFP-CD147） was successfi.dly con- structed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD 147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also signifi- cantly increased after the upregulation of CD 147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the prolif- eration and invasive ability significantly increased in the A549-CD 147 cells. Conclusion A lentiviral CD 147 expression vector and its A549 cell line （AS49-CD 14） were successfully constructed. CD 147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

关 键 词：CD147 慢病毒载体 转染 增殖 

分 类 号：R734.2[医药卫生—肿瘤]