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目的:探讨肠三叶因子对胃黏膜上皮细胞紧密连接的保护作用,并研究PI3K/Akt信号通路在其中的作用机制。方法:体外培养GES-1细胞,分别设正常对照组,LPS组,ITF组,LPS+ITF组,LPS+ITF+LY294002组,ITF+LY294002组。正常对照组:正常培养;LPS组:加入浓度为10mg/L的LPS;ITF组:加入浓度为100μg/L的ITF;LPS+ITF组:加入浓度为10mg/L的LPS,同时加入100μg/L的ITF;LPS+ITF+LY294002组:加入浓度为10mg/L的LPS、100μg/L的ITF,同时加入PI3K/Akt信号通路的抑制剂LY294002(15μM);ITF+LY294002组:加入100μg/L的ITF,同时加入15μM的LY294002。培养48h,采用Western blot检测ITF对PI3K/Akt信号通路的作用,采用免疫荧光和Western blot检测细胞紧密连接蛋白Occludin和ZO-1的变化情况。结果:Western blot检测结果说明,与对照组相比,ITF提高了pAkt蛋白的表达水平,而LY294002抑制了ITF激活的pAkt蛋白的表达,说明ITF可以通过激活PI3K/Akt信号通路来调控GES-1细胞的生理活动。免疫荧光和Western blot结果显示LPS导致GES-1细胞的紧密连接遭到破坏,降低紧密连接蛋白Occludin和ZO-1的表达水平,而ITF可以通过激活PI3K/Akt信号通路来保护GES-1细胞的紧密连接的完整性。结论:ITF保护胃黏膜上皮细胞紧密连接的完整性,提高紧密连接蛋白的表达水平,其发挥作用的主要分子机制是通过激活PI3K/Akt信号通路来实现的。
Objective: To investigate the protective effect of intestinal trefoil factor on gastric mucosal epithelial tight junction and investigate the mechanism of PI3K / Akt signaling pathway. Methods: GES-1 cells were cultured in vitro. Normal control group, LPS group, ITF group, LPS + ITF group, LPS + ITF + LY294002 group and ITF + LY294002 group were established. LPS group: LPS group (10mg / L); ITF group: ITF group (100μg / L); LPS + ITF group (LPS + ITF group) Of ITF; LPS + ITF + LY294002 group: adding LPS with LPS concentration of 10mg / L, ITF of 100μg / L and inhibitor of PI3K / Akt signaling pathway at 15μM; ITF + LY294002 group: adding 100μg / L ITF while adding 15 μM of LY294002. After cultured for 48h, the effect of ITF on PI3K / Akt signaling pathway was detected by Western blot. The changes of tight junction proteins Occludin and ZO-1 were detected by immunofluorescence and Western blot. Results: Western blot results showed that ITF increased pAkt protein expression compared with control group, while LY294002 inhibited ITF-activated pAkt protein expression, indicating that ITF can regulate GES-1 by activating PI3K / Akt signaling pathway Physiological activity of cells. Immunofluorescence and Western blot results showed that LPS led to the disruption of the tight junctions of GES-1 cells and decreased the expression of the tight junction proteins Occludin and ZO-1, while ITF could protect the GES-1 cells by activating the PI3K / Akt signaling pathway Closely linked integrity. Conclusion: ITF protects the integrity of tight junctions of gastric mucosal epithelial cells and enhances the expression of tight junction protein. The main molecular mechanism of ITF is through the activation of PI3K / Akt signaling pathway.