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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。
Objective To investigate the effect of SIGIRR on the expression of Toll-like receptor 5 (TLR5) induced by flagellin by up-regulating the expression of SIGIRR in human airway epithelial cell line H292. Methods The experiment was divided into 6 groups: H292 group (untreated H292 cells), eH292 group (cells transfected with empty plasmid pEGFP-N1), sH292 group (cells transfected with SIGIRR-EGFP eukaryotic expression plasmid) The above groups of cells which were added with flagellin (final concentration 0.2μg / ml) were named as FH292 group, FeH292 group and FsH292 group, respectively. The eukaryotic expression vector of SIGIRR-EGFP fusion protein was constructed. The transfected cells were transfected by lipofectamine. The expression of fusion protein was observed by fluorescence microscopy after 24h. The expression of TLR5 protein was detected by Western blotting, and the concentration of TNF-α in the supernatant was determined by ELISA. Results H292 cells transfected with recombinant vector could express SIGIRR-EGFP fusion protein, which mainly distributed in cytoplasm. The optical density of the TLR5 protein band was observed in the FH292 group compared with the H292 group (1.71 ± 0.12), the FH292 group (7.32 ± 0.99), the FH292 group (2.32 ± 0.13), the FsH292 group (7.01 ± 0.83) than sH292 group (2.01 ± 0.07) TLR5 protein expression was significantly increased (P <0.01). The concentration of TNF-α in FH292 group (114.06 ± 10.34) was significantly higher than that in H292 group (43.52 ± 2.84), while the concentration of TNF-α in FH292 group (69.56 ± 11.23) was significantly lower than that of FeH292 group (117.76 ± 9.07) (P <0.01). Conclusion Upregulation of SIGIRR can inhibit the production of TNF-α by flagellin-induced H292 cells and has no effect on the expression of TLR5. SIGIRR plays a “brake” role in this signaling pathway, but this effect is not mediated by TLR5.