蛙皮素受体激活蛋白对脂多糖诱导的人气道上皮细胞株黏液高分泌的影响
发布时间：2018-08-05 12:50
【摘要】：目的:探讨蛙皮素受体激活蛋白(BRAP)对脂多糖(LPS)诱导的气道黏液高分泌的影响及相关作用机制。方法:体外培养人气道上皮HBE16细胞,给予LPS刺激,构建气道黏液高分泌模型,转染已构建好的p EGFP-N1-BRAP,以空质粒载体作为对照,以ROS清除剂DMTU、ERK抑制剂U0126为干预因素。实验设空白对照组、LPS刺激组、LPS+pEGFP-N1-BRAP组、LPS+pEGFP-N1组、DMTU+LPS组、DMTU+LPS+pEGFP-N1-BRAP组、DMTU+LPS+p EGFP-N1组、U0126+LPS组、U0126+LPS+p EGFP-N1-BRAP组、U0126+LPS+pEGFP-N1组。MTT法检测细胞活性;RT-PCR法检测黏蛋白(MUC)5AC转录水平;ELISA法检测MUC5AC、IL-1β和IL-6蛋白水平;Western blot法检测BRAP、p-IΚBα、p-ERK、p-p38MAPK蛋白水平;测定活性氧(ROS)含量;激光共聚焦法检测MUC5AC蛋白表达水平。结果:(1)给予LPS刺激后,细胞MUC5ACmRNA及蛋白水平明显增高,ROS含量和炎症因子IL-1β、IL-6的表达量也明显增加,同时伴有p-ERK、p-p38MAPK和p-IΚBα蛋白表达增高(P值均0.01);(2)细胞过表达BRAP后,MUC5ACmRNA及蛋白水平、ROS含量、炎症因子(IL-1β、IL-6)表达量和p-ERK及p-IΚBα蛋白水平均明显降低(P值均0.01);(3)与U0126+LPS组和空质粒对照处理组相比,U0126+LPS+p EGFP-N1-BRAP组细胞MUC5AC转录及蛋白水平降低(P0.05),同时p-ERK和p-IΚB蛋白表达也降低(P0.01)。(4)在DMTU+LPS+p EGFP-N1-BRAP组,MUC5AC转录及蛋白水平较DMTU+LPS组和空质粒对照处理组显著降低(P0.05),ROS含量降低(P0.01),p-ERK及p-IΚBα蛋白水平呈同样的降低趋势(P0.01)。结论:BRAP过表达可以通过抑制ROS/ERK/NF-ΚB信号通路,降低LPS诱导的气道黏蛋白MUC5AC高分泌。
[Abstract]:Aim: to investigate the effect of bombesin receptor activator protein (BRAP) on airway mucus hypersecretion induced by lipopolysaccharide (LPS) and its related mechanism. Methods: human airway epithelial HBE16 cells were cultured in vitro and stimulated with LPS. The airway mucus hypersecretion model was constructed and transfected into pEGFP-N1-BRAP. empty plasmid vector was used as control and U0126 as ROS scavenger inhibitor U0126 as intervention factor. The experiment was divided into two groups: LPS-stimulated group (LPS-stimulated group), lipopolysaccharide group (pEGFP-N1-BRAP group), lipopolysaccharide group (LPS group), DMTU LPS p EGFP-N1 group (U0126 LPS group) and U0126 LPS p EGFP-N1-BRAP group (U0126 LPS pEGFP-N1 group). MTT assay was used to detect cellular activity, RT-PCR, RT-PCR and Elisa for detection of MUC5ACU IL-1 尾 and IL-6 egg. Western blot assay was used to detect the protein level of BRAPP p-I 尾 -B 伪 -ERKP p-p38 MAPK. The content of reactive oxygen species (ROS) and the expression of MUC5AC protein were detected by confocal laser scanning. Results: (1) after stimulation with LPS, the levels of MUC5ACmRNA and protein increased significantly, the content of Ros and the expression of IL-1 尾 -IL-6, the inflammatory factor IL-1 尾 -IL-6, were also increased, and the expression of p-ERK p-p38 MAPK and p-I K B 伪 protein was increased (P all 0.01); (2), and the Ros content of MUC5AC mRNA and protein was also increased after BRAP overexpression. The expression of IL-1 尾 -IL-6 and the levels of p-ERK and p-I K B 伪 protein were significantly lower than those of U0126 LPS p EGFP-N1-BRAP group and U0126 LPS p EGFP-N1-BRAP group (P < 0.05). The expression of p-ERK and p-I K B protein was also decreased in U0126 LPS p EGFP-N1-BRAP group (P < 0.05), and the expression of p-ERK and p-I K B protein was also decreased in U0126 LPS p EGFP-N1-BRAP group (P < 0.05). (P0.01). (4) the levels of MUC5AC transcription and protein in DMTU LPS p EGFP-N1-BRAP group were significantly lower than those in DMTU LPS group and blank plasmid control group (P0.05). Conclusion the overexpression of BRAP can decrease the secretion of airway mucin MUC5AC induced by LPS by inhibiting the signal pathway of Ross / ERK / NF-KB.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R56
本文编号：2165849
[Abstract]:Aim: to investigate the effect of bombesin receptor activator protein (BRAP) on airway mucus hypersecretion induced by lipopolysaccharide (LPS) and its related mechanism. Methods: human airway epithelial HBE16 cells were cultured in vitro and stimulated with LPS. The airway mucus hypersecretion model was constructed and transfected into pEGFP-N1-BRAP. empty plasmid vector was used as control and U0126 as ROS scavenger inhibitor U0126 as intervention factor. The experiment was divided into two groups: LPS-stimulated group (LPS-stimulated group), lipopolysaccharide group (pEGFP-N1-BRAP group), lipopolysaccharide group (LPS group), DMTU LPS p EGFP-N1 group (U0126 LPS group) and U0126 LPS p EGFP-N1-BRAP group (U0126 LPS pEGFP-N1 group). MTT assay was used to detect cellular activity, RT-PCR, RT-PCR and Elisa for detection of MUC5ACU IL-1 尾 and IL-6 egg. Western blot assay was used to detect the protein level of BRAPP p-I 尾 -B 伪 -ERKP p-p38 MAPK. The content of reactive oxygen species (ROS) and the expression of MUC5AC protein were detected by confocal laser scanning. Results: (1) after stimulation with LPS, the levels of MUC5ACmRNA and protein increased significantly, the content of Ros and the expression of IL-1 尾 -IL-6, the inflammatory factor IL-1 尾 -IL-6, were also increased, and the expression of p-ERK p-p38 MAPK and p-I K B 伪 protein was increased (P all 0.01); (2), and the Ros content of MUC5AC mRNA and protein was also increased after BRAP overexpression. The expression of IL-1 尾 -IL-6 and the levels of p-ERK and p-I K B 伪 protein were significantly lower than those of U0126 LPS p EGFP-N1-BRAP group and U0126 LPS p EGFP-N1-BRAP group (P < 0.05). The expression of p-ERK and p-I K B protein was also decreased in U0126 LPS p EGFP-N1-BRAP group (P < 0.05), and the expression of p-ERK and p-I K B protein was also decreased in U0126 LPS p EGFP-N1-BRAP group (P < 0.05). (P0.01). (4) the levels of MUC5AC transcription and protein in DMTU LPS p EGFP-N1-BRAP group were significantly lower than those in DMTU LPS group and blank plasmid control group (P0.05). Conclusion the overexpression of BRAP can decrease the secretion of airway mucin MUC5AC induced by LPS by inhibiting the signal pathway of Ross / ERK / NF-KB.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R56
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