特异性shRNA抑制人近端肾小管上皮细胞Toll样受体4的表达

Specific shRNA Suppress the Expression of Toll Like Receptor 4 in Human Proximal Tubular Epithelial Cells

目的:研究RNA干扰法对人近端肾小管上皮细胞(HKC)中Toll样受体4(TLR4)表达的抑制作用。方法:将针对TLR4分子设计的采用体外转录生物合成的特异性短发卡RNA(shRNA)双链分子用脂质体转染的方法导入体外培养的HKC;应用免疫荧光染色在共聚焦显微镜下观察TLR4的分布及表达强弱;半定量RT-PCR和免疫蛋白印迹检测TLR4及内参照-βactin在mRNA水平和蛋白水平的表达量。结果:干扰组TLR4的荧光强度显著低于对照组,其mRNA和蛋白的表达量分别下降了67%和62%。同时观察到TLR4主要分布于HKC细胞的胞膜及胞质区。结论:采用生物体外转录的方法成功合成针对TLR4的shRNA双链分子,并有效地转入HKC细胞中使TLR4的表达下调。

Objective： To investigate the role of suppression on Toll like receptor 4 （TLR4） of cultured human proximal tubular epithelial cells （HKCs） with RNA interference（RNAi）. Methods： Firstly, the short hairpin RNA（shRNA） which specifically targeting TLR4 mRNA was biologic synthesized with in vitro transcription, then was transfected into cultured HKC. The distribution levels of TLR4 were revealed by immunofluorescence staining under laser scanning confocal microscopy. The mRNA and protein expression levels of TLR4 and β-actin were detected by semiquantitative RT-PCR and Western blotting. Results. The fluorescence intensity of TLR4 was sig- nificantly lower than that in control group. The reduction of the mRNA expression in TLR4 was apparently detected by about 67~ compared to control group. The protein expression levels of TLR4 decreased significantly by about 62 % compared to control group. The distribution of TLR4 was mainly located on cell membrane and cytoplasmic domain. Conclusion： The specific shRNA duplex molecule targeting TLR4 mRNA was successfully synthesized and efficiently transfected into HKCs. The expression of TLR4 of HKCs was suppressed by RNAi.