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

doi:10.3760/cma.j.issn.1673-422X.2017.10.001

摘要/Abstract

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Western blotting检测AMPK的表达情况，四甲基偶氮唑蓝（MTT）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）×105∶(3.4±0.3)×105，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。

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

Abstract: ObjectiveTo 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. MethodsFull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 supernatant to infect the A549 cells and establish a stably, overexpressed cell line A549. The mRNA and protein of AMPK were examined by realtime quantitative 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. ResultsGV358AMPK lentivirus vectors was successfully constructed by restrictive enzyme digestion and plasmid 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. Meanwhile, 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)×105 vs. (3.4±0.3)×105, P=0.004］ of A549 cells. ConclusionThe lentiviral AMPK expression vector and its A549 cell line is successfully constructed. AMPK overexpression inhibits the proliferation and invasive ability of A549 cells.

Key words: Carcinoma, nonsmallcell lung, Lentivirus, Cell proliferation, AMPactivated protein kinase

张相民，刘联斌，曾汶，周茂华，叶桂林，叶永强，王刚，李韶今. 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[J]. 国际肿瘤学杂志, 2017, 44(10): 721-726.

Zhang Xiangmin, Liu Lianbin, Zeng Wen, Zhou Maohua, Ye Guilin, Ye Yongqiang, Wang Gang, Li Shaojin. Construction of overexpression lentiviral vector and its expression in lung cancer A549 cells of AMPactivated protein kinase[J]. Journal of International Oncology, 2017, 44(10): 721-726.

参考文献

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