發(fā)布時間：2018-08-05 12:50

【摘要】：目的:探討蛙皮素受體激活蛋白(BRAP)對脂多糖(LPS)誘導的氣道黏液高分泌的影響及相關(guān)作用機制。方法:體外培養(yǎng)人氣道上皮HBE16細胞,給予LPS刺激,構(gòu)建氣道黏液高分泌模型,轉(zhuǎn)染已構(gòu)建好的p EGFP-N1-BRAP,以空質(zhì)粒載體作為對照,以ROS清除劑DMTU、ERK抑制劑U0126為干預因素。實驗設(shè)空白對照組、LPS刺激組、LPS+pEGFP-N1-BRAP組、LPS+pEGFP-N1組、DMTU+LPS組、DMTU+LPS+pEGFP-N1-BRAP組、DMTU+LPS+p EGFP-N1組、U0126+LPS組、U0126+LPS+p EGFP-N1-BRAP組、U0126+LPS+pEGFP-N1組。MTT法檢測細胞活性;RT-PCR法檢測黏蛋白(MUC)5AC轉(zhuǎn)錄水平;ELISA法檢測MUC5AC、IL-1β和IL-6蛋白水平;Western blot法檢測BRAP、p-IΚBα、p-ERK、p-p38MAPK蛋白水平;測定活性氧(ROS)含量;激光共聚焦法檢測MUC5AC蛋白表達水平。結(jié)果:(1)給予LPS刺激后,細胞MUC5ACmRNA及蛋白水平明顯增高,ROS含量和炎癥因子IL-1β、IL-6的表達量也明顯增加,同時伴有p-ERK、p-p38MAPK和p-IΚBα蛋白表達增高(P值均0.01);(2)細胞過表達BRAP后,MUC5ACmRNA及蛋白水平、ROS含量、炎癥因子(IL-1β、IL-6)表達量和p-ERK及p-IΚBα蛋白水平均明顯降低(P值均0.01);(3)與U0126+LPS組和空質(zhì)粒對照處理組相比,U0126+LPS+p EGFP-N1-BRAP組細胞MUC5AC轉(zhuǎn)錄及蛋白水平降低(P0.05),同時p-ERK和p-IΚB蛋白表達也降低(P0.01)。(4)在DMTU+LPS+p EGFP-N1-BRAP組,MUC5AC轉(zhuǎn)錄及蛋白水平較DMTU+LPS組和空質(zhì)粒對照處理組顯著降低(P0.05),ROS含量降低(P0.01),p-ERK及p-IΚBα蛋白水平呈同樣的降低趨勢(P0.01)。結(jié)論:BRAP過表達可以通過抑制ROS/ERK/NF-ΚB信號通路,降低LPS誘導的氣道黏蛋白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 偽 -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 偽 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 偽 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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R56

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本文編號：2165849