摘要
目的:探讨一氧化氮(NO)对肠上皮细胞表达紧密连接蛋白Occludin的影响,以研究NO对肠黏膜屏障的作用机制.方法:将NO的供体Sin1与肠上皮细胞株Caco-2共培养24 h,采用MTT方法观察NO对肠上皮细胞的作用,并分别提取细胞蛋白和总RNA,采用免疫蛋白印迹(Westem blot)蛋白半定量方法和实时定量聚合酶链式反应(RO-PCR)方法检测不同NO浓度对Caco-2细胞表达紧密连接蛋白Occludin蛋白和mRNA表达的影响.结果:随着Sin1浓度升高(125,250,500和1000μmol/L)NO对细胞的杀伤作用产生并逐渐增大,Occludin蛋白表达量和mRNA的相对表达量与无Sin1刺激时蛋白及mRNA的表达量相比明显降低(蛋白:375±0.5,374±0.8,363±0.3.363±0.7 vs 398±0.7;mRNA:0.689±0.01,0.578±0.09,0.554±0.03,0.619±0.04 vs 1,均P<0.01).结论:NO可直接损伤肠上皮细胞,同时以剂量依赖形式在蛋白和分子水平影响紧密连接蛋白Occludin的表达.
AIM： To study the effects of NO on the intestinal mucosal barrier and on the tight junction protein occludin in intestinal epithelial cells in vitro. METHODS： Colon cancer cell line （Caco-2 cells） was treated with Sin1, a NO donor, in a dose-dependent manner for 24 hours. The protein and total RNA of Caco-2 cells were extracted. Changes in occludin protein mRNA in Caco-2 cells stimulated by NO were determined by Westem blotting and real-time quantitative pclymerase chain reaction, respectively. RESULTS： The killing effect of NO on Caco-2 cells was dose-dependent. When treated with Sin1 at 125, 250, 500 and 1000 ,mol/L doses, the levels of occludin protein （375 ± 0.5, 374 ± 0.8, 363 ± 0.3, 363 ± 0.7） and mRNA （0.689 ± 0.01, 0.578 ± 0.09, 0.554 ± 0.03, 0.619 ± 0.04） were significantly decreased compared with those in untreated Caco-2 cells （398 ± 0.7, 1, respectively, P 〈 0.01）.CONCLUSION： NO can directly kill intestinal epithelial cells. NO may affect protein and mRNA expression of the tight junction protein occludin in a dose-dependent manner.
出处
《世界华人消化杂志》
CAS
北大核心
2007年第21期2295-2299,共5页
World Chinese Journal of Digestology
