中华胃肠外科杂志
中華胃腸外科雜誌
중화위장외과잡지
CHINESE JOURNAL OF GASTROINTESTINAL SURGERY
2013年
5期
479-483
,共5页
杨洋%王文生%邱远%孙力华%杨桦
楊洋%王文生%邱遠%孫力華%楊樺
양양%왕문생%구원%손력화%양화
缺氧%环磷酸腺苷%肠黏膜屏障%紧密连接蛋白
缺氧%環燐痠腺苷%腸黏膜屏障%緊密連接蛋白
결양%배린산선감%장점막병장%긴밀련접단백
Hypoxia%Cyclic adenosine monophosphate%Intestinal barrier function%Tight junctions
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Western blot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occludin mRNA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occludin mRNA及蛋白水平的表达,从而减轻缺氧对肠上皮屏障的损伤.
目的 探討缺氧條件下環燐痠腺苷(cAMP)水平的變化對腸黏膜屏障功能的影響.方法 採用人結腸癌Caco-2細胞建立腸上皮細胞缺氧模型,分為常氧組、常氧加Forskolin組、缺氧8h組和缺氧8h加SQ22536(腺苷痠環化酶抑製劑)組.採用cAMP檢測試劑盒,檢測實驗各組cAMP水平的變化;RT-PCR及Western blot方法分彆檢測各組腸上皮緊密連接蛋白claudin-1和occludin的mRNA和蛋白錶達水平的變化；Millipore電阻抗繫統法檢測各組腸上皮細胞跨上皮電阻(TER)的變化情況.結果 缺氧8h組腸上皮cAMP含量較常氧組明顯升高[(6.30±0.50) pmol/L比(2.38±0.18) pmoL/L,P＜0.01],而claudin-1和occludin的mRNA及蛋白錶達水平均明顯降低(均P＜0.05),TER值下降瞭6.30±0.64 (P＜0.01).與缺氧8h組相比,缺氧8h加SQ22536組腸上皮cAMP含量明顯降低[(2.12±0.23) pmol/L比(6.30±0.50) pmol/L,P＜0.01],claudin-1和occludin mRNA及蛋白錶達水平明顯升高(P＜0.01),TER值升高瞭32.96±2.16(均P＜0.05).結論 缺氧條件下cAMP水平升高,而下調cAMP水平可增加腸道緊密連接蛋白claudin-1及occludin mRNA及蛋白水平的錶達,從而減輕缺氧對腸上皮屏障的損傷.
목적 탐토결양조건하배린산선감(cAMP)수평적변화대장점막병장공능적영향.방법 채용인결장암Caco-2세포건립장상피세포결양모형,분위상양조、상양가Forskolin조、결양8h조화결양8h가SQ22536(선감산배화매억제제)조.채용cAMP검측시제합,검측실험각조cAMP수평적변화;RT-PCR급Western blot방법분별검측각조장상피긴밀련접단백claudin-1화occludin적mRNA화단백표체수평적변화；Millipore전조항계통법검측각조장상피세포과상피전조(TER)적변화정황.결과 결양8h조장상피cAMP함량교상양조명현승고[(6.30±0.50) pmol/L비(2.38±0.18) pmoL/L,P＜0.01],이claudin-1화occludin적mRNA급단백표체수평균명현강저(균P＜0.05),TER치하강료6.30±0.64 (P＜0.01).여결양8h조상비,결양8h가SQ22536조장상피cAMP함량명현강저[(2.12±0.23) pmol/L비(6.30±0.50) pmol/L,P＜0.01],claudin-1화occludin mRNA급단백표체수평명현승고(P＜0.01),TER치승고료32.96±2.16(균P＜0.05).결론 결양조건하cAMP수평승고,이하조cAMP수평가증가장도긴밀련접단백claudin-1급occludin mRNA급단백수평적표체,종이감경결양대장상피병장적손상.
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 claudin-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 occludinexpression 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.
