Effects of Lipofectamine 2000/siRNA complexes on autophagy in hepatoma cells.
Effects of Lipofectamine 2000/siRNA complexes on autophagy in hepatoma cells.
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DOI：
10.1007/s12033-011-9422-6
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发表时间：
2012-05
影响因子：
2.6
通讯作者：
Shen WC
中科院分区：
文献类型：
--
作者：
Mo RH;Zaro JL;Ou JH;Shen WC
Lipofectamine 2000通常用于siRNA转染。然而，很少有研究检查细胞对这种传递系统的反应。本研究的目的是评价脂质体Lipofectamine 2000介导的siRNA转染对细胞自噬的影响。用Lipofectamine 2000/阴性对照siRNA（NC siRNA）复合物处理稳定转染以表达GFP-LC 3的Huh 7.5细胞。在处理后的不同时间点，裂解细胞并通过免疫印迹和荧光光谱法进行分析。还使用共聚焦显微镜观察细胞。在用Lipofectamine 2000/NC siRNA复合物处理的细胞中观察到内源性LC 3脂化、GFP-LC 3荧光和自噬体斑点的增加。GFP-LC 3荧光增加的动力学与转染的NC siRNA的浓度相关，其中50、100和200 nM NC siRNA分别在转染后72、48和24小时引起显著增加。对于用与两种其他NC siRNA复合的Lipofectamine 2000处理的细胞，也观察到对GFP-LC 3信号的类似作用。Lipofectamine 2000介导的NC siRNA转运导致自噬体以剂量和时间依赖性方式增加。因此，当使用这种方法进行siRNA的细胞内递送时，应考虑对细胞的这种影响。
Lipofectamine 2000 is commonly used for siRNA transfections. However, few studies have examined cellular responses to this delivery system. The purpose of this study is to evaluate the effect of siRNA transfection using Lipofectamine 2000 on cellular autophagy. Huh7.5 cells, stably-transfected to express GFP-LC3, were treated with Lipofectamine 2000/negative control siRNA (NC siRNA) complexes. At different time points after treatment, cells were lysed and analyzed by immunoblotting and fluorescence spectroscopy. Cells were also observed using confocal microscopy. An increase of endogenous LC3 lipidation, GFP-LC3 fluorescence, and autophagosomal puncta was observed in cells treated with Lipofectamine 2000/NC siRNA complexes. The kinetics of the increase of GFP-LC3 fluorescence correlated with the concentration of NC siRNA transfected, where 50, 100, and 200 nM NC siRNA caused a significant increase at 72, 48 and 24 hours, respectively, after transfection. A similar effect on the GFP-LC3 signal was also observed for cells treated with Lipofectamine 2000 complexed with two other NC siRNAs. Lipofectamine 2000-mediated transport of NC siRNAs led to an increase of autophagosomes in a dose- and time-dependent manner. Thus, this effect on cells should be taken into consideration when using this approach for intracellular delivery of siRNA.