Arkadia表达载体转染肾小管上皮细胞条件的优化

摘要

本试验旨在优化泛素化酶Arkadia质粒表达载体pGPU6/GFP/Neo转染小鼠肾小管上皮细胞(renal tubular epithelial cells,RTECs)的条件,为研究Arkadia表达载体在小鼠马兜铃酸肾病(aristolochic acid nephrohathy,AAN)中的作用奠定基础.以小鼠RTECs为转染对象、脂质体LipofectamineTM 2000为转染介质,采用Real-time PCR和CCK-8法检测不同的质粒与脂质体比例条件下Arkadia mRNA的表达差异及对细胞活性的影响；同时,用Real-time PCR检测不同的转染时间条件下表达载体对Arkadia mRNA沉默效果的差异.结果显示,Arkadia- pGPU6/GFP/Neo载体的转染效率表现出了一定的剂量和时间依赖性.当质粒DNA∶LipofectamineTM 2000为0.66 μg∶2 μL、转染时间为24 h时,对Arkadia mRNA表达的抑制最明显并且对细胞的毒性最小.最终确定了Arkadia-pGPU6/GFP/Neo载体转染小鼠RTECs的最佳条件.%To lay the foundation for investigating the role of Arkadia expression vector in mouse aristolochic acid nephropa-thy (AAN), we optimized the transfection conditions of ubiquitination enzyme Arkadia plasmid expression vector pGPU6/ GFP/Neo in mice renal tubular epithelial cells (RTECs). Mice RTECs were taken for transfection object, and liposome Lipo-fectamineTM 2000 for transfection medium, we detected the Arkadia Mrna expressions differences and the affects of cell activity under the different ratios conditions of plasmid and liposome by Real-time PCR and CCK-8 assay: at the same time, the differences of Arkadia Mrna expressions silence under the different transfection time conditions by Real-time PCR. The results showed that transfection efficiency of Arkadia-Pgpu6/GFP/Neo vector displayed dose and time dependents. When the ratio of plasmid DNA : LipofectamineTM 2000 was 0. 66 μg : 2 Μl, and the transfection time was 24 h, Arkadia Mrna expression inhibition was very obvious and the cytotoxicity was the lowest. Lastly, we determined the best transfection condition of Arkadia-Pgpu6/GFP/Neo vector.