聚乙烯亚胺介导水通道蛋白5基因表达载体体外转染的研究

Transfection of the AQP5 gene mediated by a novel cational carrier PEI in vitro

目的探讨新型阳离子载体聚乙烯亚胺(PEI)介导水通道蛋白5(AQP5)体外转染人肺腺癌细胞系A549的效果。方法将含绿色荧光蛋白的真核表达载体pEGFP-N1,以lipofectam ine2000为阳性对照,流式细胞仪检测转染效率,根据不同N/P比(5、10、15、20、25)与PEI组成转染复合物转染A549细胞,优化最适N/P比;将AQP5全长cDNA插入pEGFP-N1载体,构建pEGFP-N-AQP5,转染A549细胞,采用PCR与Western blot法检测被转染细胞中AQP5的表达。结果流式细胞仪检测GFP发光率,N/P比为15时PEI/pEGFP-N1复合物转染效率最高,可达到89.72%,与脂质体91.44%相近;应用PEI与lipofectam ine2000将pEGFP-N-AQP5成功转染A549,与空白组比较,AQP5的基因及蛋白表达均增高。结论新型阳离子载体PEI可成功介导pEGFP-N-AQP5转染A549细胞。

Objective To investigate the effect of a new cationic carrier polyethylenimine （PEI） on transfection of the aquaporin 5 （AQP5） gene into A549 cells in vitro. Methods According to different N/P ratios （5, 10, 15, 20 and 25 ）, the eukaryotic expression vector pEGFP-N1 was mixed with the green fluorescent protein（GFP） and PEI to make a transfection complex, using lipofectamine2000 as the positive control. Transfection efficiency was detected by flow cytometry for optimizing the optimal N/P ratio. Full-length AQP5 cDNA were cloned into the pEGFP-N1 vector to con- struct pEGFP-N-AQP5, which was then transfected into A549 cells. Expression of AQP5 in transfected cells was detec- ted using PCR and Western blot. Results Flow cytometry revealed the GFP emitting. When the N/P ratio was 15, the PEI/pEGFP-N1 complex achieved the highest transfection efficiency of 89.72%, close to 91.44% when delivered by liposomes. Compared with the control group, the AQP5 gene and protein expressions were significantly increased after successful transfection of pEGFP-N-AQP5 into A549 by application of the PEI and lipofectamine2000 complex. Con- clusion pEGFP-N-AQP5 can be successfully transfected into A549 cells mediated by the novel cationic carrier PEI.