促肾上腺皮质激素释放因子对肠上皮细胞紧密连接相关蛋白的调节

发布时间：2018-01-10 04:31

本文关键词：促肾上腺皮质激素释放因子对肠上皮细胞紧密连接相关蛋白的调节　出处：《世界华人消化杂志》2013年24期 　论文类型：期刊论文

【摘要】：目的:探讨促肾上腺皮质激素释放因子(corticotrophin-releasing factor,CRF)对肠上皮细胞紧密连接相关蛋白的调节作用.方法:以CRF(20 ng/mL)刺激HT-29细胞24 h后再以脂多糖(lipopolysaccharides,LPS)(100 ng/mL)刺激24 h,收集细胞,ELISA法检测细胞提取物中胞质附着蛋白(zonula occludins protein,ZO)-1、ZO-2、Occludin、cldn1、cldn2、cldn3和cldn4的表达.再次将HT-29细胞以CRF 0、1、10、20 ng/mL的浓度刺激24 h和核因子B(nuclear factor B,NF-B)抑制剂马来酸二乙酯(diethylmaleate,DEM)1 mmol/L预孵1 h后加CRF 20 ng/mL刺激24 h,然后更换各组细胞液后继续再以LPS 100 ng/mL的浓度孵育24 h,收集各组HT-29细胞,提取蛋白,免疫印迹法检测cldn2表达水平.结果:ELISA检测结果显示以CRF刺激结肠上皮细胞24 h后再以LPS刺激后ZO-1、ZO-2、Occludin、cldn1和cldn3表达水平较单纯LPS刺激有轻度的下降,但是cldn2的表达则较前明显升高(P0.01).免疫印迹法检测结果显示以LPS孵育前以CRF(1 ng/mL)刺激即可引起cldn2表达增高,随后加大CRF刺激浓度,分别以10和20 ng/mL刺激,则cldn2的表达随着刺激浓度加大而逐渐增高(P0.05),呈现剂量依赖效应,而以CRF(20 ng/mL)刺激同时加入NF-B抑制剂DEM后再以LPS孵育HT-29细胞后,CRF则不能诱导cldn2表达增高(P0.05).结论:CRF对肠上皮细胞中的cldn2表达刺激呈剂量依赖效应,并且CRF对肠上皮细胞的这种诱导作用可能与NF-B激活有关.
[Abstract]:Objective: To explore the effect of corticotropin releasing factor (corticotrophin-releasing, factor, CRF) of the intestinal epithelial tight junction protein regulation. Methods: CRF (20 ng/mL) 24 h after the stimulation of HT-29 cells with lipopolysaccharide (lipopolysaccharides, LPS) (100 ng/mL) stimulation of 24 h, a collection of cells, cell extracts detection ELISA in cytoplasmic attachment protein (zonula occludins protein, ZO) -1, ZO-2, Occludin, CLDN1, cldn2, cldn3 and cldn4. The expression of HT-29 cells in a concentration of CRF to 0,1,10,20 ng/mL stimulation 24 h and nuclear factor B (nuclear factor B, NF-B) inhibitor maleic acid ethyl ester two (Diethylmaleate, DEM) 1 mmol/L 1 h after preincubation with CRF 20 ng/mL 24 h stimulation, then replace the cells in each group after incubation with liquid concentration to LPS 100 with ng/mL 24 h, HT-29 cells were collected, extracted the protein level of cldn2 expression was detected by Western blot. Results: ELIS A results showed that colonic epithelial cells stimulated by CRF 24 After h after stimulation with LPS ZO-1, ZO-2, Occludin, CLDN1 and cldn3 expression compared to LPS stimulation with slight decline, but the expression of cldn2 was significantly higher than before (P0.01). Western blot results showed that incubation with LPS in CRF (1 ng/mL) stimulation can induce the expression of cldn2, then increase the concentration of CRF, respectively to 10 and 20 ng/mL stimulation, cldn2 expression gradually increased with increasing stimulus concentration (P0.05), showed a dose dependent effect, with CRF (20 ng/mL) stimulation while adding NF-B inhibitor DEM after incubation with LPS HT-29 cells, CRF can not induce increased expression of cldn2 (P0.05). Conclusion: the expression of CRF in a dose dependent stimulation of intestinal epithelial cells in cldn2, and the CRF of intestinal epithelial cells induced by NF-B may be related to activation.

【作者单位】：郑州人民医院消化内科;郑州大学第二附属医院消化科;
【分类号】：R363
【正文快照】： 背景资料很多肠道疾病特别是炎症性肠病都与肠黏膜屏障损伤有关,而肠上皮毗邻细胞之间形成紧密连接的屏障需要紧密连接相关蛋白的作用,已经证实应激因素与多种肠病的发生有关,但是否与肠黏膜屏障的破坏有关尚不清楚.同行评议者刘占举,教授,同济大学附属上海市第十人民医院核

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