SIGIRR对鞭毛蛋白诱导的H292细胞TLR5表达的影响

摘要

Objective To explore the effects of SIGIRR on the flagellin-induced expression of Toll-like receptor 5 (TLR5) in human air-way epithelial cell H292. Methods Cells were divided into 6 groups: H292 group, cells without any treatment; eH292 group, cells were transfected with empty plasmid pEGFP-N1; sH292 group, cells were transfected with recombinant plasmid SIGIRR-EOFP. In the other three groups, FH292 group, FeH292 group and FsH292 group, cells were additionally treated with flagellin (final concentration of 0. 2μg/ml). The eukaryotic expression vector of fusion protein SIGIRR-EGFP was constructed, and then tranafected into H292 cells by liposome transfection. The expression of fusion protdn was observed with fluorescence microscope 24 hours after transfection, the concentration of TNF-α ha supematant was detected by ELISA assay, and the expression of TLR5 protein was detected by Western blotting, Results The fusion protein SIGIRR-EGFP was expressed and mainly distributed in the plasma of H292 cell transfected with the recombinant vectors. The OD value of TLR5 protein was higher in FH292 group (8. 06～ 1.53) than in H292 group (1. 71±0. 12), higher in FeH292 group (7. 32±0. 99) than in eH292 group (2. 32±0. 13), and also higher in FsH292 group (7. 01-4-0. 83) than in sH292 group (2. 01±0. 07, P<0. 01). The concentration of TNF-α increased significantly (P< 0. 01) in FH292 group (114. 06 ～ 10. 34) than in H292 group (43. 52 ± 2. 84), while decreased significantly (P<0.01) in FaH292 group (69.56±11.23) than in H292 group (117. 76±9.07). Condmions Up-regulated expression of SIGIRR may inhibit the flagellin-induced release of TNF-α, however it shows no effect on the expression of TLRS. It seems that the SIGIRR serves as a "brake" on TLRs signaling, but it is not induced by TLR5.%目的 通过上调SIGIRR在人气道上皮细胞株H292中的表达,研究SIGIRR对鞭毛蛋白诱导的Toll样受体5(TLR5)表达的影响.方法 实验设以下6组:H292组(未给予任何处理的H292细胞)、eH292组(转染空质粒pEGFP-N1的细胞)、sH292组(转染SIGIRR-EGFP真核表达质粒的细胞),另设加入鞭毛蛋白(终浓度0.2μg/ml)的上述各组细胞,分别命名为FH292组、FeH292组和FsH292组.构建SIGIRR-EGFP融和蛋白的真核表达载体,脂质体转染法转染细胞,24h后用荧光显微镜观察融合蛋白的表达情况.Western blotting检测TLR5蛋白表达,ELISA法测定细胞上清中TNF-α浓度.结果 转染重组载体的H292细胞可表达SIGIRR-EGFP融和蛋白,其主要分布于细胞质.通过对TLR5蛋白条带的光密度进行观察发现,FH292组(8.06±1.53)较H292组(1.71±0.12)、FeH292组(7.32±0.99)较eH292组(2.32±0.13)、FsH292组(7.01±0.83)较sH292组(2.01±0.07)TLR5蛋白表达明显增加(P<0.01).FH292组TNF-α浓度(114.06±10.34)较H292组(43.52±2.84)明显增加,而FsH292组TNF-α浓度(69.56±11,23)较FeH292组(117.76±9.07)显著下降(P<0.01).结论 SIGIRR的表达上调可抑制鞭毛蛋白诱导的H292细胞中TNF-α的产生,对TLR5表达无影响.SIGIRR在该信号通路中发挥了"刹车"作用,但该作用并不是由TLR5介导的.