利用慢病毒载体系统构建稳定表达癌基因PIN1的鼻咽上皮细胞株

Establishment of the stable PIN1-over-expressed cell lines in NP69 cells by letiviral system

目的利用慢病毒载体系统构建稳定表达癌基因肽脯氨酰异构酶PIN1的鼻咽上皮细胞株,用于后续生物学功能方面研究。方法将病毒载体p CDH-PIN1用Lipofectamine转染到293TN细胞,收集含有PIN1病毒颗粒的培养液加入NP69细胞进行培养。荧光显微镜观察慢病毒载体GFP发光率、RT-q PCR、Westernblot验证PIN1的表达情况。结果荧光显微镜观察发现PIN1基因已整合到94%±2.09%的NP69细胞中,转染效果稳定。RT-q PCR、Westernblot检测发现NP69-PIN1组中PIN1的mRNA和蛋白水平明显高于对照组和空白组。结论课题组利用慢病毒载体系统成功在鼻咽上皮细胞构建PIN1过表达株。

Objective Using lentiviral system to establish the stable PIN1-over-expressed cell lines in NP69 cells. Methods The p CDH lentiviral vector was packed using the 3rd generation lentivirus system according to manufacturer＇s protocol. Using Lipofectamine^TM2000,293 TN cells were transfected with p CDH-PIN1 vector.Viral supernatant（ culture media） was harvested and added into NP69 cell for co-culture. GFP reporter expression was confirmed under the fluorescence microscope. PIN1 expression was confirmed by quantitative polymerase chain reaction（ RT-q PCR） and western blotting assay. Results GFP reporter expressions were observed in94% ± 2. 09% NP69 cells by fluorescent microscope. Significant increases in the expressions of both PIN1 transcript and protein were detected in the stable PIN1 expressing NP69 compared with those infected with vector control and parental cells. Conclusion Lentiviral system is successfully used to establish the stable PIN1-over-expressed cell lines in NP69 cells.