摘要目的 探讨细菌鞭毛蛋A对肠黏膜屏障影响及肥大细胞在肠黏膜屏障损伤中作用.方法 在Transwell小室内共同培养T84细胞单层和肥大细胞(HMC-1),模拟肠黏膜屏障,并向培养系统中加入细菌鞭毛蛋白.通过测量不同条件下T84细胞单层的跨上皮电阻(TER)值和辣根过氧化酶(HRP)通过率,分析肠黏膜屏障完整性;使用免疫细胞化学实验检测T84细胞对细菌鞭毛蛋白的摄取;使用酶联免疫吸附实验(ELISA)检测基底层培养基中经T84细胞转运的细菌鞭毛蛋白含量,以及HMC-1细胞分泌的组胺和类胰蛋白酶β2(MCT)的水平.结果 较低浓度(≤8mg/L)的细菌鞭毛蛋白对T84细胞单层的相对TER值以及HRP通过率无显著影响;当细菌鞭毛蛋白浓度>8mg/L,随着细菌鞭毛蛋白浓度的增加T84细胞单层的相对TER值逐渐下降,HRP通过率逐渐增加.免疫细胞化学结果显示,和正常对照组相比,细菌鞭毛蛋白组T84细胞的胞质被染成棕褐色.ELISA结果提示细菌鞭毛蛋白组基底侧细菌鞭毛蛋白吸光度A值明显高于正常对照组,并且各组组胺浓度和MCT水平与各组细菌鞭毛蛋白HRP通过率的结果保持一致.结论 细菌鞭毛蛋白可直接破坏T84细胞单层或经T84细胞摄取并转运到达基底侧,激活HMC-1细胞,使其释放组胺及类胰蛋白酶β2,增强T84细胞单层肠黏膜屏障的损伤.

abstractsObjectives To investigate the effect of bacterial flamellin on intestinal mucosal barrier and the role of mast cells in the injury of intestinal mucosal barrier. Methods Single layer of T84 cells and mast cells(HMC-1) were co-cultured in Transwell chamber to simulate intestinal mucosal barrier. and different concentrations of bacterial flamellin were added to this culture system. The integrity of intestinal mucosal barrier was evaluated by measuring the value of transepithelial electric resistance (TER) and the passing rate of horseradish peroxidase (HRP) in above culture system. Cell immunocytochemistry assay was used to investigate the uptake of bacterial flagellin by T84 cells. The amount of transported bacterial flagellin by T84 and the level of histamine and tryptase β2 (MCT) secreted by HMC-1 were detected by enzyme-linked immunosorbent assay (ELISA). Results There were no significant changes of TER value and HRP passing rate when T84 cells were stimulated with lower concentration of bacterial flagellin (≤8 mg/L) in the culture system. However. the TER value decreased and HRP passing rate increased gradually with the increase of bacterial flagellin concentration (> 8 mg/L). Immunocytochemical results showed that the cytoplasm of T84 cells was dyed brown when compared with normal controls. ELISA results revealed that the A value of the basolateral flagellin in flagellin group was significantly higher than that in normal control group. Furthermore. the histamine concentrations and MCT levels in each group were consistent with the results of the flagellin HRP passing rate in each group. Conclusion Bacterial flagellin can directly destroy T84 cell-monolayer. or can be transported by T84 cells to the basolateral side. then can stimulate HMC-1 cells to release histamine and MCT. enhancing the damage of intestinal mucosal barrier.