同型半胱氨酸影响Caco-2细胞通透性的机制研究  

Mechanisms of homocysteine on Caco-2 cell permeability

作　　者：姚成云 丁少桢 杨青[1] 刘晓昌[1] 徐张巍[1] 梅俏[1] 许建明[1] Yao Chengyun;Ding Shaozhen;Yang Qing(Dept of Gastroenterology,The First Affiliated Hospital of Anhui Medical University,Hefei 230022)

机构地区：[1]安徽医科大学第一附属医院消化内科

出　　处：《安徽医科大学学报》2020年第1期95-99,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81470809)

摘　　要：目的探讨同型半胱氨酸(Hcy)对Caco-2细胞通透性的影响及其机制。方法 Caco-2结肠癌细胞分为空白对照组、Hcy处理组(10、20、50μmol/L)、Hcy+不同抑制剂处理组(ERK抑制剂PD98059、MLCK抑制剂ML-7、P38MAPK抑制剂SB203580、Ca2+/Calmodulin抑制剂KN62、Rho抑制剂Y27632及PKC抑制剂Staurosporine)。通过检测跨上皮电流阻抗(TEER)及异硫氰酸荧光素(FITC)跨膜转运量评估Caco-2细胞模型通透性,Western blot法检测Caco-2细胞中MEK、ERK、MLCK、p-ERK、p-MLCK、ZO-1、Occludin、Claudin-1及Claudin-2蛋白表达水平。结果与空白对照组相比,使用50μmol/L Hcy预处理的Caco-2细胞通透性改变速率最快。Claudin-1、Occludin、ZO-1表达降低,MEK、ERK、MLCK、p-ERK、p-MLCK、Claudin-2蛋白表达升高。与Hcy处理组相比,使用ML-7及PD98059处理Caco-2细胞,可减弱Hcy预处理引起的Caco-2细胞通透性增加,降低MEK、ERK、MLCK、p-ERK、p-MLCK、Claudin-2蛋白表达,提高Claudin-1、Occludin及ZO-1蛋白表达。结论 Hcy可能通过MEK-ERK-MLCK细胞信号通路,调控细胞紧密连接(TJ)相关蛋白表达,影响Caco-2细胞通透性。Objective To investigate the effect of homocysteine(Hcy) on the permeability of Caco-2 cells and its mechanism. Methods Caco-2 cells were divided into blank control group,treated with different concentrations of Hcy(10, 20, 50 μmol/L), treated with Hcy + different signaling transduction inhibitors(ERK inhibitor PD98059, MLCK inhibitor ML-7, P38 MAPK inhibitor SB203580, Ca2+/Calmodulin inhibitor KN62, Rho inhibitor Y27632 and PKC inhibitor Staurosporine). The permeability of Caco-2 cells was detected by transepithelial electrical resistance(TEER) and fluorescein isothiocyanate(FITC). The expression levels of MEK, ERK, MLCK, p-ERK, p-MLCK, ZO-1, Occludin, Claudin-1 and Claudin-2 in Caco-2 cells were detected by Western blot. Results Compared with the blank control group, the permeability of Caco-2 cells pretreated with 50 μmol/L Hcy had the faster change. The expression of Claudin-1, Occludin and ZO-1 decreased, while the expression of MEK, ERK, MLCK, p-ERK, p-MLCK and Claudin-2 increased. Compared with Hcy group, pretreatment with ML-7 and PD98059 could reduce the permeability alteration of Caco-2 cells induced by Hcy, which resulted in decreased expression of MEK, ERK, MLCK, p-ERK, p-MLCK, Claudin-2 while increased expression of Claudin-1, Occludin and ZO-1. Conclusion Hcy may regulate the expression of tight junction-related proteins through MEK-ERK-MLCK signaling pathway and affect the permeability of Caco-2 cells.

关 键 词：同型半胱氨酸 通透性 肌球蛋白轻链激酶 紧密连接 

分 类 号：R574.5[医药卫生—消化系统] R333.3[医药卫生—内科学]