缺氧条件下环磷酸腺苷对肠黏膜屏障功能的调控研究  

Role of cyclic adenosine monophosphate（cAMP） in the regulation of intestinal epithelial barrier function under hypoxia

作　　者：杨洋[1] 王文生[1] 邱远[1] 孙力华[1] 杨桦[1] 

机构地区：[1]第三军医大学新桥医院普通外科,重庆400037

出　　处：《中华胃肠外科杂志》2013年第5期479-483,共5页Chinese Journal of Gastrointestinal Surgery

基　　金：国家自然科学基金（81020108023,81272078）;重庆市科委国际合作重点项目（201110008）

摘　　要：目的探讨缺氧条件下环磷酸腺苷（cAMP）水平的变化对肠黏膜屏障功能的影响。方法采用人结肠癌Caco．2细胞建立肠上皮细胞缺氧模型，分为常氧组、常氧加Forskolin组、缺氧8h组和缺氧8h加SQ22536（腺苷酸环化酶抑制剂）组。采用cAMP检测试剂盒，检测实验各组cAMP水平的变化；RT-PCR及Westernblot方法分别检测各组肠上皮紧密连接蛋白claudin-1和occludin的mRNA和蛋白表达水平的变化：Millipore电阻抗系统法检测各组肠上皮细胞跨上皮电阻（TER）的变化情况。结果缺氧8h组肠上皮cAMP含量较常氧组明显升高[（6．30±0．50）pmol／L比（2．38±0．18）pmol／L，P〈0．01]，而claudin．1和occludin的mRNA及蛋白表达水平均明显降低（均P〈0．05），TER值下降了6．30±0．64（P〈0．01）。与缺氧8h组相比，缺氧8h加SQ22536组肠上皮cAMP含量明显降低[（2．12±0．23）pmol／L比（6．30±0．50）pmol／L，P〈0．01]，claudin-1和occludinmRNA及蛋白表达水平明显升高（P〈0．01），TER值升高了32．96±2．16（均P〈0．05）。结论缺氧条件下cAMP水平升高，而下调cAMP水平可增加肠道紧密连接蛋白claudin-1及occludinmRNA及蛋白水平的表达，从而减轻缺氧对肠上皮屏障的损伤。Objective To investigate the role of cyclic adenosine monophosphate （cAMP） in the regulation of intestinal epithelial barrier function under hypoxia. Methods Intestinal epithelial barrier was established by Caco-2 monolayers. Cells were divided into four groups： normoxia （Nx）, normoxia plus Forskolin （Nx±FSK）, hypoxia （Hx）, hypoxia plus SQ22536 （Hx±SQ22536）. cAMP concentrations of different groups were assessed by cAMP enzyme immunoassay kit. RT-PCR and Western blotting were used to detect the mRNA and protein expressions of clandin-1 and occludin under normoxic and hypoxic condition. Caco-2 monolayers were grown on Millicell filters, and transepithelial electrical resistance （TER） was measured using a Millipore electric resistance system. Results The concentration of cAMP under hypoxic conditions （Hx group） was higher compared with Nx group [（6.30±0.50） pmol/L vs. （2.38±0.18） pmol/L, P〈0.01 ]. At the same time, both mRNA and protein expressions of claudin-1 and occluding were lower in Hx group than those in Nx group （all P〈0.05）. TER decreased by 76.30±0.64 （P〈0.01）. When the monolayers were exposed to hypoxia plus SQ22536 （Hx＋SQ22536 group）, the concentration of cAMP was（2.12±0.23） pmol/L, which was lower than that under hypoxic conditions （Hx group, P〈0.01）. Both mRNA and protein expressions of claudin-1 and occludin were higher compared to Hx group （all P〈0.01）. TER increased by 32.96±2.16 （P〈0.05）. Conclusion When Caco-2 cells are exposed to hypoxia, barrier function, claudin-1 and occludin expression are diminished in parallel with a high level of intracellular cAMP compared with the normoxic condition. Inhibition of the intracellular cAMP level under hypoxia can maintain the intestinal epithelial function through regulating the claudin-1 and occludin expression and attenuate the permeability of intestinal mucosa.

关 键 词：缺氧 环磷酸腺苷 肠黏膜屏障 紧密连接蛋白 

分 类 号：R285.5[医药卫生—中药学]