利用慢病毒载体构建稳定干扰AMPKα1的HeLa细胞系

摘要

目的 建立稳定干扰AMPKα1的HeLa细胞系.方法 利用AMPKα1慢病毒干扰质粒PLKO.1-puro-AMPKα1转染293T细胞制备重组慢病毒,然后用重组慢病毒感染HeLa细胞,荧光显微镜观察病毒感染效率,免疫印迹实验检测Sh-AMPKα1病毒感染组的AMPKα1表达.然后采用嘌呤霉素筛选出稳定干扰AMPKα1的HeLa细胞单克隆,免疫印迹实验和免疫荧光实验检测AMPKα1表达情况.最后用AMPK激活剂Metformin实验验证AMPKα1稳定干扰的Hela细胞中AMPKα1蛋白的表达以及AMPKα1的生物功能.结果 荧光拍照结果显示慢病毒成功感染Hela细胞;免疫印迹实验显示特异性Sh-AMPKα1病毒感染组的AMPKα1表达[(0.58±0.02)DPI]比WT组[(1.00±0.00)DPI]低,免疫荧光实验结果也显示Sh-AMPKα1病毒感染组中AMPKα1的平均光密度[(0.09±0.01)IOD/area]比WT组[(1.00±0.00)IOD/area]要低,差异均具有显著统计学意义(P<0.01);经AMPK激活剂Metformin处理后,AMPKα1稳定干扰的Hela细胞中仍无AMPKα1蛋白表达,并且Sh-AMPKα1组中LC3-Ⅱ/Ⅰ/Actin[(1.00±0.00I)DPI]也显著低于HA-AMPKα1组[(1.62±0.02)DPI],表明病毒感染组细胞中AMPKα1不能发挥其生物功能,差异具有显著统计学意义(P<0.01).结论 利用ShRNA-AMPKα1慢病毒筛选出了高效干扰AMPKα1表达的HeLa细胞系,为后续深入研究AMPKα1的生物功能奠定了基础.%Objective To establish adenosine monophosphate-activated protein kinaseα1 (AMPKα1) stably in-terfered Hela cell line. Methods Lentiviral vector PLKO.1-puro-AMPKα1 was transfected into 293T cells to prepare recombinant lentivirus. Then HeLa cells were infected with the recombinant lentivirus, and the efficiency of virus infec-tion was detected by fluorescent photography. The monoclonal Hela cell stably interfered AMPKα1 was screened by pu-romycin, and Western blot and immunofluorescence were used to detecte the expression of AMPKα1 in specific Sh-AMPKα1 virus infected group. Finally, the biological function of AMPKα1 in Hela cells stably interfered with AMPKα1 under the treatment of metformin, a AMPK activator, was detected. Results The results of fluorescent pho-tography showed that the virus infection was highly efficient. Immunoblotting results showed that the expression of AMPKα1 was significantly reduced in Hela cells stably interfered with AMPKα1 (0.58 ± 0.02) DPI compared with the WT group (1.00 ± 0.00) DPI. Immunofluorescence results also showed that the mean optical density of AMPKα1 was (0.09±0.01) IOD/area in Sh-AMPKα1 virus infected group versus (1.00±0.00) IOD/area in the WT group. All the above differences were statistically significant (P<0.01). After the treatment of metformin, the expression of AMPKα1 in the Sh-AMPKα1 virus infected group was still not expressed. Moreover, the relative ratio of LC3-Ⅱ/Ⅰ/Actinin in the Sh-AMPKα1 group was (1.00 ± 0.00) DPI, which was significantly lower than (1.62 ± 0.02) DPI in the HA-AMPKα1 group, indicating that AMPKα1 could not play its biological function (P<0.01). Conclusion The above results revealed that AMPKα1 effectively interfered Hela cell line was established by ShRNA-AMPKα1 lentivirus, which laid the foun-dation for the further research of AMPKα1 biological function.