AMPK过表达慢病毒载体的构建及稳定转染肺癌A549细胞株的建立  

Construction of overexpression lentiviral vector and its expression in lung cancer A549 cells of AMP-activated protein kinase

作　　者：张相民 刘联斌[1,2] 曾汶 周茂华 叶桂林 叶永强[1,2] 王刚 李韶今 

机构地区：[1]赣南医学院第一临床医学院,赣州341000 [2]赣南医学院附属肿瘤医院肿瘤防治研究所

出　　处：《国际肿瘤学杂志》2017年第10期721-726,共6页Journal of International Oncology

基　　金：国家自然科学基金（81441069）

摘　　要：目的通过构建表达载体，建立稳定转染腺苷酸活化蛋白激酶（AMPK）的肺癌A549细胞株，观察过表达AMPK后对A549细胞增殖、侵袭能力的影响。方法扩增AMPK全长序列，双酶切目的基因并插入GV358载体，共转染293T细胞进行慢病毒包装，收集慢病毒上清液后感染A549细胞，建立稳定过表达AMPK的A549细胞株。实时荧光定量PCR（qRT—PCR）和Westernblotting检测AMPK的表达情况，四甲基偶氮唑蓝（MTF）及Transwell法检测A549细胞增殖、侵袭能力的变化。结果经限制内切酶鉴定及测序分析，成功构建了GV358．AMPK慢病毒表达载体质粒。与转染空载体相比，转染GV358慢病毒载体的A549细胞AMPK蛋白表达升高5．87倍（P=0．002），mRNA水平升高16．12倍（P〈0．001）；A549细胞过表达AMPK后，第4-5天细胞增殖活性显著降低（第4天：0．53±0．03：0．64±0．05，P=0．021；第5天：0．58±0．04：0．80±0．07，P=0．002），侵袭能力显著减弱[（1．6±0．5）×10^5：（3．4±0．3）×10^5，P=0．004]。结论成功构建了AMPK慢病毒表达载体和稳定表达A549细胞株，过表达AMPK可抑制A549细胞增殖及侵袭能力。Objective To establish a stable lung cancer A549 cell line transfected by AMP-activated protein kinase （AMPK） expression vector, and to observe the effect of AMPK on proliferation as well as on the invasive ability of A549 cells. Methods Full-length of AMPK gene was amplified and its target gene was digested, then inserted into the GV358 plasmid. Co-tranfected 293T cells were subjected to the lentivirus equipment package. Subsequently, we collected the lentivirus supematant to infect the A549 cells and establish a stably, overexpressed cell line A549. The mRNA and protein of AMPK were examined by real-time quantita- tive reverse transcriptase polymerase chain reaction （qRT-PCR） and Western blotting. The proliferation and invasion abilities of A549 cells were detected by methyl thiazolyl thiazolium （MTT） and Transwell assay. Results GV358-AMPK lentivirus vectors was successfully constructed by restrictive enzyme digestion and plas- mid sequencing. There were significantly increased expressions of AMPK protein （5.87 times, P = 0.002） and mRNA （ 16.12 times, P 〈 0. 001 ） after transfected with GV358-AMPK compared with the Vector group. Mean- while, AMPK overexpression showed significantly lower proliferation （ the forth day： 0.53±0.03 vs. 0. 64 ±0.05, P = 0. 021 ; the fifth day： 0. 58 ±0.04 vs. 0. 80±0. 07, P = 0. 002） and weaken invasive ability [（1.6±0.5） ×10^5 vs. （3.4±0.3） ×10^5, P=0.004] of A549 cells. Conclusion The lentiviral AMPK expression vector and its A549 cell line is successfully constructed. AMPK overexpression inhibits the prolifera- tion and invasive ability of A549 cells.

关 键 词：癌 非小细胞肺 慢病毒属 细胞增殖 腺苷酸活化蛋白激酶 

分 类 号：R734.2[医药卫生—肿瘤]