PTEN shRNA慢病毒载体构建及转染人子宫腺肌病细胞的稳定细胞株筛选

Construction of Lentiviral Vector Carrying PTEN shRNA and Its Transfection into Human Adenomyosis Cell Lines in vitro

目的构建携带绿色荧光蛋白(green fluorescent protein,GFP)的第10染色体丢失的磷酸酶基因(phasphatase and tensin homo-log deleted in chromosome 10,PTEN)的短发卡RNA(short hairpin RNA,shRNA)慢病毒载体,并在转染人子宫腺肌病(adenomyosis,AM)细胞后筛选最优稳转株。方法设计合成3条特异性针对人PTEN基因的shRNA序列,构建到pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO载体中,测序鉴定载体构建情况,进而包装病毒并检测滴度。筛选出最优感染复数(multiplicity of infection,MOI)、嘌呤霉素杀灭浓度后,用慢病毒液转染AM细胞,以嘌呤霉素药筛建立PTEN敲低的稳定细胞株,采用实时定量聚合酶链反应(Real-time quantitative polymerase chain reaction,RT-qPCR)检测转染后细胞内PTEN mRNA表达水平,蛋白免疫印迹法(Western Blot,WB)检测PTEN蛋白表达水平,以检验转染效率并筛选出干扰效率最佳的shRNA序列。结果成功构建了3个PTEN shRNA慢病毒载体,包装后以MOI为50转染AM细胞,荧光显微镜观察绿色荧光表达明显,嘌呤霉素持续药筛建立了PTEN敲低的稳定细胞株,以PTEN shRNA3慢病毒载体的干扰效率最佳(92.98%)。结论成功构建了PTEN shRNA慢病毒载体,并在体外有效转染了AM细胞,建立了PTEN敲低的稳定细胞株。

Objective To construct a recombinant lentiviral vector that co-expressing green fluorescent protein and PTEN shRNA and establish an adenomyosis cell line with stable PTEN down-regulation.Methods Three interfering sequences targeting PTEN were designed and inserted into the lentiviral vector pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO.After identification by DNA sequencing,the lentiviral vectors carrying PTENshRNA were packaged and their titers were detected.The lentiviral particles were collected to infect human adenomyosis cells with screened multiplicity of infection(MOI),and adenomyosis cells with stable PTEN down-regulation were screened with puromycin.The interference efficiency was assessed by real-time PCR and Western Blot.Results DNA sequencing demonstrated successful construction of three PTENshRNA lentiviral vectors.Adenomyosis cells were infected with MOI of 50,and the transfection efficiency was observed by green florescence under fluorescence microscope.Real-time PCR and Western Blot both showed that the highest interference efficiency of PTEN shRNA3 vector of 92.98%,which resulted in obvious down-regulation of PTEN in adenomyosis cells.Conclusion This study has successfully constructed lentiviral vector of PTEN shRNA and established an adenomyosis cell model with stable PTEN down-regulation in vitro.