脂多糖促进人近端肾小管上皮细胞TLR4的表达

目的:研究人近端肾小管上皮细胞(HKC)在脂多糖(LPS)刺激下Toll样受体4(TLR4)的表达及作用。方法:实验细胞分两组,LPS刺激组(50mg/LLPS加入体外培养的HKC)和正常对照组;应用免疫荧光染色在激光共聚焦显微镜下观察LPS刺激24h后TLR4在HKC中的分布及表达强弱;半定量RT-PCR和免疫蛋白印迹检测各组TLR4及内参照β-actin在mRNA水平和蛋白水平的表达量;Annexin V-FITC结合流式细胞仪检测各组细胞的早期凋亡率情况。结果:LPS刺激组TLR4的免疫荧光强度显著高于正常对照组,TLR4主要分布于HKC的胞膜及胞质区;刺激组TLR4 mRNA表达高于正常对照组(1.051±0.082比0.38±0.036),差异有显著性(P<0.01);刺激组TLR4蛋白表达高于正常对照组(0.371±0.033比0.105±0.008),差异有显著性(P<0.01);LPS刺激组细胞的早期凋亡率(41.29%)显著高于正常对照组(2.36%)。结论:LPS能刺激HKC高表达TLR4,且促进HKC的早期凋亡,TLR4可能在LPS诱导的HKC凋亡中发挥一定的作用。

Lipopolysaccharide Increases the Expression of TLR4 in Human Proximal Renal Tubular Cells

Objective:To investigate the effect of lipopolysaccharide (LPS) on Toll like receptor 4 (TLR4) mRNA and protein expression in cultured human proximal renal tubular epithelial cells (HKCs). Methods: The cultured HKCs were divided into two groups as LPS induced group which were treated with 50 mg/L LPS for 24 hours and HKCs normal control group. The distribution levels of TLR4 in HKC were revealed by immunofluorescence staining under laser scanning confocal microscope. The mRNA and protein expression of TLR4 and β-actin was detected by semi-quantitative RT-PCR and Western blotting respectively. Apoptosis of HKCs was detected by Annexin V-FITC combined with flow cytometry. Results: The fluorescence intensity of TLR4 of LPS group was significantly higher than that in control group. The expression of TLR4 was mainly located on cell membrane and cytoplasm. The expression of TLR4 mRNA and protein in LPS group was apparently higher than that in control group respectively (1.051±0.082 vs 0.38±0.036, and 0.371±0.033 vs 0.105±0.008,P<0.01 resectively). The rate of earlier period apoptosis cells in LPS group (41.29%) was higher than that in control group (2.36%). Conclusion: LPS increases the expression of TLR4 and promotes the rate of earlier period apoptosis HKCs. TLR4 may play a role in LPS-induced HKC apoptosis.