鞭毛蛋白活化TLR5信号途径对于SPC-A-1细胞生物学行为的影响

Influence of activation of TLR5 signaling pathway stimulated by Flagellin on the biological action of SPC-A-1 cells

目的 探讨外源性配体鞭毛蛋白活化TLR5后,对于SPC-A-1细胞生物学行为的影响.方法 选用SPC-A-1细胞作为研究对象,鞭毛蛋白作用浓度为0.1μg/ml.构建TLR5-shRNA,并转染SPC-A-1细胞.实验分为四组：对照组、TLR5-shRNA组、Flagellin组和TLR5-shRNA＋ Flagellin组.用四甲基偶氮唑蓝比色法（MTT）在0、24、48、72 h检测细胞增殖情况,在48 h分别用TUNEL法检测细胞凋亡,Transwell细胞侵袭实验研究细胞侵袭能力的影响,平板划线培养实验研究细胞迁移能力的影响,细胞集落形成实验研究细胞克隆形成能力.结果 （1）于0、24、48、72 h在570 nm波长处检测各组细胞的光密度值,结果显示,在同一时间点,Flagellin组与其它三组相比,细胞增殖明显受到抑制（P＜0.01）,TLR5-shRNA＋ Flagellin组与对照组和TLR5-shRNA组相比,细胞增殖无明显影响（P＞0.05）.（2）对照组、TLR5-shRNA组、Flagellin组和TLR5-shRNA＋ Flagellin组的细胞凋亡率分别为（14.75 ±0.072）％、（15.09 ±0.053）％、（21.63 ±0.047）％、（14.98 ±0.036）％.与对照组、TLR5-shRNA组和TLR5-shRNA＋ Flagellin组相比,Flagellin组细胞凋亡率明显增加（P＜0.05）.（3）对照组、TLR5-shRNA组、Flagellin组和TLR5-shRNA＋ Flagellin组穿膜细胞数分别为39.55±2.31、42.78±3.52、18.75±3.77和38.43±2.98.与对照组、TLR5-shRNA组和TLR5-shRNA＋ Flagellin组相比,Flagellin组的细胞其侵袭能力明显受到抑制（P＜0.01）,而对照组、TLR5-shRNA组和TLR5-shRNA＋ Flagellin组两两比较,差异均无统计学意义（P＞0.05）.（4）与对照组、TLR5-shRNA组和TLR5-shRNA＋ Flagellin组相比,Flagellin刺激48 h后,Flagellin组细胞的迁移能力显著下降（P＜0.05）,而与对照组、TLR5-shRNA组相比,TLR5-shRNA＋ Flagellin组细胞的迁移能力无明显改变（P＞0.05）.（5）对照组、TLR5-shRNA组、Flagellin组和TLR5-shRNA＋ Flagellin组细胞集落形成率分别为（40.37±3.88）％、（39.88±2.49）％、（13.29±3.76）％、（38.81±4.11）％.与对照组、TLR5-shR-NA组和TLR5-shRNA＋ Flagellin组相比,Flagellin组细胞集落形成率显著减少（P＜0.01）,与对照组、TLR5-shRNA组相比,TLR5-shRNA＋ Flagellin组细胞集落率差异无统计学意义（P＞0.05）.结论 外源性配体鞭毛蛋白活化TLR5信号通路后,可以抑制SPC-A-1细胞的增殖、转移、侵袭,促进其凋亡,可以作为潜在的治疗靶点.

Objective To investigate the influence of the activation of Toll-like receptors （TLR） 5 signaling pathway stimulated by flagellin on the biological action of SPC-A-1 cells.Methods SPC-A-1 cells were chosen as the research object, and the concentration of flagellin was 0.1 μg/ml.Four groups were control, TLR5-shRNA, Flagellin, and TLR5-shRNA ＋ Flagellin groups.Cell proliferation was detected with methyl thiazolyl tetrazolium （MTT） at 0 h, 24 h, 48 h and 72 h, cell apoptosis with TdT-mediated dUTP nick-end labeling （TUNEL） assay, cell invasion with Transwell cell invasion assay, cell migration with impact streak plate culture experiments, and cell colony formation with cell colony formation experimental at 48 h.Results （1）At 0 h, 24 h, 48 h and 72 h, the optical density values was detected under 570 nm wavelength.At the same time point, compared to the other groups, the cell proliferation of the Flagellin group was significantly inhibited （P 〈 0.01）.Compared to control and TLR5-shRNA groups,there was no significant effect in cell proliferation of TLR5-shRNA ＋ Flagellin group （P 〉 0.05）.（2） The apoptosis rates of the control, TLR5-shRNA, Flagellin, and TLR5-shRNA ＋ Flagellin groups were （14.75 ±0.072）%, （15.09 ±0.053）%, （21.63% ±0.047）% and （14.98 ±0.036）%, respectively.Compared to the other groups, the apoptosis rate of the Flagellin group was significantly increased （P 〈 0.05）.（3）The number of penetrating cells of the control, TLR5-shRNA, Flagellin, and TLR5-shRNA ＋ Flagellin was 39.55 ± 2.31, 42.78 ± 3.52, 18.75 ± 3.77 and 38.43 ± 2.98, respectively.Compared to the other groups, cell invasion of the Flagellin group was significantly inhibited （P 〈 0.01） , while pairwise comparisons of the control, TLR5-shRNA, and TLR5-shRNA ＋ Flagellin groups, there was significant difference （P 〉 0.05）.（4）Compared to the other groups, cell migration of Flagellin group was significantly declined after 48h stimulated by Flagellin （P 〈0.05）.Compared to control group and TLR5-shRNA group, while the migration ability of TLR5-shRNA ＋ Flagellin group did not change significantly （P 〉0.05）.（5） The colony formation rates of the control, TLR5-shRNA, Flagellin, and TLR5-shRNA ＋ Flagellin groups were （40.37 ±3.88）%, （39.88 ±2.49）%, （13.29±3.76）%, and （38.81±4.11）%, respectively.Compared to the other groups, the colony formation of the Flagellin group was significantly decreased （P 〈 0.01）.Compared to the control and TLR5-shRNA groups, the colony formation of TLR5-shRNA ＋ Flagellin group wasn＆#39;t significantly different （P 〉 0.05）.Conclusions The activation of TLR5 signaling pathway stimulated by flagellin can inhibit proliferation, metastasis, and invasion of SPC-A-1 cells, promotes apoptosis, and might be used as potential therapeutic targets.