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  

Effect of corticotropinreleasing factor on expression of tight junction proteins in intestinal epithelial cells

作　　者：杨丽[1] 郑鹏远[2] 刘志强[2] 

机构地区：[1]郑州人民医院消化内科,河南省郑州市450003 [2]郑州大学第二附属医院消化科,河南省郑州市450003

出　　处：《世界华人消化杂志》2013年第24期2424-2428,共5页World Chinese Journal of Digestology

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(P<0.01).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(P<0.05),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(P>0.05).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.AIM： To investigate the effect of corticotropin- releasing factor on the expression of tight junc- tion proteins in intestinal epithelial cells. METHODS： HT-29 cells pretreated with corti- cotrophin-releasing factor （CRF） at a concentra- tion of 20 ng/mL for 24 h were stimulated with LPS at a concentration of 100 ng/mL for 24 h, and cellular extracts were harvested to measure the expression of ZO-1, ZO-2, Occludin, cldnl, cldn2, cldn3 and cldn4 by enzyme-linked im- munosorbent assay （ELISA）. Subsequently, groups of HT-29 cells were stimulated with CRF at a concentration of 0, 1, 10 or 20 ng/mL for 24 h, or pretreated with nuclear factor κB （NF-~：B） inhibitor diethylmaleate （DEM, 1 mmol/L） andthen incubated with CRF at a concentration of 20 ng/mL for 24 h. Each group was then stimu- lated with LPS at a concentration of 100 ng/mL for 24 h, and the expression of cldn2 protein was detected by Western blot. RESULTS： The expression of ZO-1, ZO-2, Occlu- din, cldnl, cldn3 and cldn4 was changed slightly （all P 〉 0.05） while the expression of cldn2 was markedly increased （P 〈 0.05） in HT-29 cells after exposure to both LPS and CRF. CRF sig- nificantly increased the expression of cldn2 in HT-29 cells in a dose-dependent manner, while NF-κB inhibitor DEM could block this effect. CONCLUSION： CRF significantly increases the expression of cldn2 in HT-29 cells in a dose- dependent manner, and NF-κB activation is involved in CRF-induced cldn2 expression. CRF has no effect on the expression of ZO-1, ZO-2, Occludin, cldnl, cldn3 and cldn4 in HT-29 cells.

关 键 词：促肾上腺皮质激素释放因子 肠上皮细胞 紧密连接 脂多糖 

分 类 号：R363[医药卫生—病理学]