TLR-4信号转导通路介导层流切应力诱导血管内皮细胞IL-8基因表达的研究

Involvement of TLR-4 signaling pathway in shear stress induced IL-8 gene expression in ECV304 cells

目的 :观察TLR - 4信号转导通路在层流低切应力诱导血管内皮细胞IL - 8基因表达中的作用。方法 :设计突变引物 ,用RT -PCR技术从血管内皮细胞扩增出胞内区段缺失突变TLR4cDNA ,用PCR技术从其DNA中扩增出IL - 8上游调控序列 (IL - 8USCS) ,分别克隆于真核表达质粒pcDNA3及绿色荧光增强蛋白报告基因pEGFP1质粒 ,构建出重组TLR - 4缺失突变基因真核表达质粒pcDNA3 -mTLR4和IL - 8报告基因表达质粒pEGFP1-IL8USCS。用pEGFP1 -IL8USCS转染或pcDNA3 -mTLR4和pEGFP1 -IL8USCS共转染ECV30 4细胞 ,用 4 2dyne/cm2 层流切应力刺激 3h ,流式细胞仪观察荧光蛋白表达强度变化。细胞裂解物IκB免疫印迹和NF -κBp65免疫荧光细胞化学染色检测IκB的磷酸化及降解和NF -κB的活化。结果 :用pEGFP1 -IL8USCS转染细胞 ,经层流切应力刺激 3h后荧光蛋白表达增强 (1 0 6∶2 71 ) ,同样用pcDNA3 -mTLR4和pEGFP 1 -IL8USCS共转染细胞 ,层流切应力刺激 3h后荧光蛋白表达未明显增强。细胞裂解物免疫印迹显示 ,刺激 1 0min时磷酸化IκB即显著增强 ,1h后磷酸化IκB印迹强度几乎降到基线水平 ,而IκB随刺激时间延长而逐渐降低 ,1h后几乎测不到IκB。NF -κBp65免疫荧光细胞化学染色显示 ,切应力刺激 0 5h ,胞核即出现阳性反应 ,刺?

AIM: To examine the role of TLR-4 signaling pathway in laminar low shear stress-induced IL-8 gene expression in ECV304 cells. METHODS: RT-PCR and PCR were used to amplify a TLR4 mutant (lacking the 155 COOH terminal amino acids of the wild type TLR4 ) and-102-+61 bp 5′-flanking region of IL-8 gene (IL8 USCS)from endothelial cells. These two DNA fragments were cloned into pcDNA3 and pEGFP1, respectively, and the recombinant plasmid pcDNA3-mTLR4 and pEGFP1-IL8USCS were obtained. ECV304 cells were transfected with the pEGFP1-IL8USCS or co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 by using Dosper liposomal transfectional reagent and selected by G418, and then stimulated by 4 2 dyne/cm 2 shear stress for 3 hours. The green fluorescent protein expression was analyzed by Flow Cytometry. Immunoblotting of the cell lysates and NF-κB p65 immunocytofluorescent staining were used to determine IκB phosphorylation, degradation and NF-κB activation. RESULTS: Flow Cytometric analysis showed that when exposed to 4 2 dyne/cm 2 shear stress for 3 hours, there was a marked increase in the enhanced green fluorescent protein expression in pEGFP1-IL8USCS-transfected ECV304 cells. In contrast, there was almost no change in the green fluorescent protein expression in the cells co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 after the stimulation. Western blot analysis showed that a markedly increased p-IκB of cell lysates occurred at 10 min of exposure and the blot density almost dropped down to the baseline after 60 min of exposure. The density of IκB blot dropped down with increasing exposure time. NF-κB p65 immunocytofluorescent staining of ECV304 cells showed that when exposed to the same flow shear stress for 0 5,1 hours, the cell nuclei became staining, and after 1 5 or 2 hours, the staining was very strong. CONCLUSION: These results suggested that the inflammatory TLR-4/NF-κB signaling pathway may be involved in flow shear stress-induced IL-8 gene expression in human vascular endothelial cells.