摘要
目的建立稳定干扰AMPKα1的He La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He La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He La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Sh RNA-AMPKα1慢病毒筛选出了高效干扰AMPKα1表达的He La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 kinase α1（AMPKα1） stably in-terfered Hela cell line. Methods Lentiviral vector PLKO.1-puro-AMPKα1 was transfected into 293 T cells to prepare re-combinant lentivirus. Then He La cells were infected with the recombinant lentivirus, and the efficiency of virus infectionwas detected by fluorescent photography. The monoclonal Hela cell stably interfered AMPKα1 was screened by puromycin,and Western blot and immunofluorescence were used to detecte the expression of AMPKα1 in specific Sh-AMPKα1 virusinfected group. Finally, the biological function of AMPKα1 in Hela cells stably interfered with AMPKα1 under the treat-ment of metformin, a AMPK activator, was detected. Results The results of fluorescent photography showed that the virusinfection was highly efficient. Immunoblotting results showed that the expression of AMPKα1 was significantly reduced inHela cells stably interfered with AMPKα1（0.58±0.02） DPI compared with the WT group（1.00±0.00） DPI. Immunofluores-cence results also showed that the mean optical density of AMPKα1 was（0.09±0.01） IOD/area in Sh-AMPKα1 virus infect-ed group versus（1.00±0.00） IOD/area in the WT group. All the above differences were statistically significant（P〈0.01）. Af-ter 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 significantlylower 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 Sh R-NA-AMPKα1 lentivirus, which laid the foundation for the further research of AMPKα1 biological function.
出处
《海南医学》
CAS
2017年第23期3785-3789,共5页
Hainan Medical Journal
基金
国家自然科学基金(编号:31401182)
广东省自然科学基金(编号:2014A030313533)
广东省科技发展专项资金(编号:2016A020215152)
广东省粤东西北地区引进紧缺拔尖人才"扬帆计划"人才项目(编号:4YF14007G)
广东医科大学科研基金(编号:M2014024
M2015001)
关键词
慢病毒载体
AMPKα1
RNA干扰
HELA细胞
METFORMIN
Lentiviral vector
Adenosine monophosphate-activated protein kinase al （AMPKczl）
RNA interfer-ence
HeLa cells
Metformin
