摘要
目的在piggy Bac(PB)转座子介导下,探索在HepG2细胞中实现多个药物代谢酶稳定共表达的策略,为建立体外药物代谢和肝毒性研究的理想细胞模型奠定基础。方法首先选择3种目前针对大片段DNA使用较广的转染方法:Lipofectamine~?LTX,Gen Jet^(TM)(Ver.Ⅱ)和Neon~?Transfection System,分别转染N端标记增强型绿色荧光蛋白质粒(pEGFP-N2)和2A串联重组细胞色素3A4(CYP3A4)和CYP2C19的PB转座子质粒(pPB-CYP3A4-2A-2C19)至HepG2细胞,48 h后比较不同方法的转染效率和细胞毒性,确立高效低毒的HepG2细胞转染方法。然后选择该法进行转染,将设计的3组PB重组转座子:单基因转座子(PB-CYP3A4)、2A串联多基因转座子(PB-CYP3A4-2A-2C19)、多个单基因转座子混合物[PB-CYP3A4,PB-CYP2C8,PB-CYP2A6,有机阴离子转运多肽1B1的PB转座子(PB-OATP1B1)]分别转染HepG2细胞,利用嘌呤霉素和GFP进行单克隆细胞筛选:挑选具有抗性且表达GFP的细胞单克隆并进行扩大培养,采用实时荧光定量PCR、Western蛋白质印迹和高效液相色谱-串联质谱联用法分别检测各组药物代谢酶m RNA、蛋白质水平及酶活性,并进行统计分析。结果 3种转染方法比较发现,Gen Jet^(TM)法的转染效率显著高于其他2种(P<0.01),介导pEGFP-N2和pPB-CYP3A4-2A-2C19的转染效率分别高达(94.2±2.5)%和(89.3±3.3)%,且该法具有较低的细胞毒性。因此选择Gen Jet^(TM)法进行后续PB转座子转染。分别转染3组PB重组转座子发现,单个转座子PB-CYP3A4和PB-CYP3A4-2A-2C19转染后,筛选获得的抗性细胞克隆中各个药物代谢酶在m RNA、蛋白质及活性水平均显著提高,其中2A可实现CYP3A4和CYP2C19药物代谢酶协同稳定表达;而多个转座子混合共转染细胞中,仅有随机几个基因表达;且各药物代谢酶基因的表达不均衡,仅CYP3A4药物代谢酶基因在单克隆细胞中表达水平明显升高。结论 Gen Jet^(TM)法可成为PB重组转座子转染HepG2细胞的有效方法。PB转座子可介导基因稳定表达;但在实现多基因共表达时,与病毒载体法不同,理论上可行的PB多转座子共转染方法其基因表达效率下降,且各基因表达不均衡。相比之下,应用2A串联多个基因串联重组PB单转座子可实现各基因稳定表达。
OBJECTIVE To study the methodology of achieving stable co-expression of drug-metabolizing enzymes in the HepG2 cells by the piggy Bac（PB） transposon system. METHODS N-terminal attachment of enhanced green fluorscent protein plasmid（pEGFP-N2） and 2 A peptide linked recombinant PB transposon plasmid containing dual-genes encoding drug metabolizing enzymes cytochrome P450 3 A4（CYP3A4） and CYP2 C19（pPB-CYP3A4-2 A-2 C19） were transfected into HepG2 cells respectively by LipofectamineLTX reagent, GenJet（TM）（Ver.Ⅱ） reagent and NeonTransfection System reagent, which were widely used for large-sized DNA fragments transfection. 48 h later, the transfection efficiency and cell toxicity were detected and compared between the three methods so as to find a method with relatively high efficiency and low toxicity for later transfection. Then, three groups of recombinant PB transposons-single-gene transposon（PB-CYP3A4）, 2 A peptide linked dual-gene transposon（PB-CYP3A4-2 A-2 C19） and multiple single-gene transposon mixture[PB-CYP3A4, PBCYP2 C8, PB-CYP2 A6, organic anion transporting polypeptide 1 B1 PB transposon（PB-OATP1 B1）]-were transfected into HepG2 cells respectively with the above established method. The puromycin（Puro）-resistant and GFP positive cell clones were picked up and further cultured. The mRNA, protein and metabolic levels of drug-metabolizing enzymes in monoclonal cell lines were detected by quantitative real-time PCR, Western blotting and high performance liquid chromatography-tandem mass spectrometry respectively after screening by Puro and green fluorescence. Comparisons of different groups were made using statistical analysis. RESULTS The comparison of three different transfection methods indicated that the transfection efficiency of GenJet^TM was up to（94.2±2.5）% and（89.3±3.3）%, significantly higher than those of the other two methods（P0.01）, along with lower cytotoxicity. Then GenJet^TM was chosen for later transfection. In the Puro-resistant monoclonal cell lines of single transposon PBCYP3A4, PB-CYP3A4-2 A-2 C19 groups, the mRNA, protein and enzyme activity levels of drug-metabolizing enzymes were significantly increased respectively. The recombinant transposon（PB-CYP3A4-2 A-2 C19） containing 2 A peptide could achieve stable and efficient co-expression of two metabolizing enzymes CYP3A4 and CYP2 C19, while the expression of drug-metabolizing enzymes remained unbalanced and random in those of multiple single-gene transposon mixture group（PB-CYP3A4, PBCYP2 C8, PB-CYP2 A6, PB-OATP 1 B1）（CYP3A4 was expressed in some cell clones only）. CONCLUSION GenJet^TM could be an effective method for the PB recombinant transposon transfection into HepG2 cells, by which the PB transposon could mediate stable expression of drug-metabolizing enzymes. In terms of multi-gene expression, a low and unbalanced expression is found by multiple transposons cotransfection method, which is different from that by virus mediated method. In contrast, mono-PB transposon linked by 2 A peptide can achieve stable expression of multi-genes.
作者
庞士慧
钟国瑞
谢水林
李浩健
邹淑香
贾英
戴仁科
黄黎珍
PANG Shi-hui;ZHONG Guo-rui;XIE Shui-lin;LI Hao-jian;ZOU Shu-xiang;JIA Ying;DAI Ren-ke;HUANG Li-zhen(School of Bioscience and Bioengineering;Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China)
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2018年第2期125-134,共10页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金项目(81202585)
广东省科技计划项目(2014A030304014)~~
作者简介
庞士慧，硕士研究生，主要从事药物肝毒性研究。;通讯作者：黄黎珍，E-mail：huanglzh@scut．edu．cn，Tel：13631406992
