Yu H T, Lu P R
Department of Ophthalmology, First Affiliated Hospital of Soochow University, Soochow, Jiangsu, China.
Department of Ophthalmology, Northern Jiangsu People's Hospital, Yangzhou, Jiangsu, China.
Genet Mol Res. 2016 Jun 3;15(2):gmr7897. doi: 10.4238/gmr.15027897.
The aim of this study was to observe the proliferation of, and cell-cycle changes in, the human lens epithelial cell line HLEC after Toll-like receptor 4 (TLR4) gene silencing. HLEC cells were transfected with four TLR4-short hairpin RNA (shRNA) lentiviral vectors or the control lentivirus (pGCL-GFP-shRP-1, -2, -3, -4, NC). TLR4 silencing was verified in these cells 96 h post-transfection using real-time polymerase chain reaction and western blot. We also observed the change in number of pGCL-GFP-shRP-4-transfected HLEC cells with silenced TLR4 (multiplicity of infection = 10). Cell proliferation was analyzed 48 h after transfection by a standard Cell Counting Kit-8 (CCK-8) assay, and the cell cycle changes were detected by flow cytometry. The number of cells with silenced TLR4 decreased with time. The decrease in TLR4 expression led to decelerated cell proliferation. Cells with silenced TLR4 (for 48 h) were arrested in the G1 phase; that is, the cell cycle was prolonged and cell division was decelerated. Lentivirus-mediated RNA interference effectively silenced TLR4 expression in HLEC cells, which decelerated their proliferation rate and extended the cell cycle.
本研究旨在观察Toll样受体4（TLR4）基因沉默后人晶状体上皮细胞系HLEC的增殖及细胞周期变化。用四种TLR4短发夹RNA（shRNA）慢病毒载体或对照慢病毒（pGCL-GFP-shRP-1、-2、-3、-4、NC）转染HLEC细胞。转染后96小时，使用实时聚合酶链反应和蛋白质印迹法在这些细胞中验证TLR4沉默。我们还观察了TLR4沉默的pGCL-GFP-shRP-4转染HLEC细胞数量的变化（感染复数=10）。转染后48小时，通过标准细胞计数试剂盒-8（CCK-8）测定法分析细胞增殖，并通过流式细胞术检测细胞周期变化。TLR4沉默的细胞数量随时间减少。TLR4表达的降低导致细胞增殖减速。TLR4沉默的细胞（48小时）停滞在G1期；即细胞周期延长，细胞分裂减速。慢病毒介导的RNA干扰有效沉默了HLEC细胞中TLR4的表达，从而降低了其增殖速率并延长了细胞周期。
