缺氧条件下血红素氧合酶-1对肠黏膜屏障的保护作用

Protective effect of heme oxygenase-1 on intestinal epithelial barrier function of Caco-2 cells under hypoxia

目的 观察缺氧条件下血红素氧合酶-1（HO-1）对肠黏膜屏障功能的影响.方法 HO-1真核过表达载体转染Caeo-2细胞,分为常氧组、缺氧组、转染组、质粒对照组.实时定量聚合酶链反应（Real-time PCR）、Western blot方法检测肠上皮细胞紧密连接蛋白Occludin和HO-1的mRNA和蛋白的表达水平变化.免疫荧光法观察Occludin在细胞内的分布.结果 与常氧组比较,缺氧组Occludin的mRNA（0.43±0.03比1.0,P＜0.01）、蛋白（0.62±0.07比1.0,P＜0.01）表达水平均明显降低;与缺氧组比较,转染组Occludin的mRNA（0.65 ±0.04比0.43 ±0.03,P＜0.05）、蛋白（0.89±0.06比0.62 ±0.07,P＜0.05）表达水平明显升高;缺氧组与质粒对照组Occludin的mRNA（0.43±0.03比0.40±0.05,P＞0.05）、蛋白（0.62 ±0.07比0.61 ±0.04,P＞0.05）表达无明显变化.免疫荧光染色见Occludin沿细胞膜分布,呈线性荧光,缺氧引起其形态破坏,转染HO-1后破坏减轻.结论 缺氧条件下,HO-1可增加肠道紧密连接蛋白Occludin的表达水平,减轻缺氧对肠黏膜屏障的损伤.

Objective To investigate the regulator effect of heme oxygenase-1 （HO-1） on intestinal epithelial barrier function under hypoxia.Methods Eukaryotic overexpression vector pShuttle-CMVHO-1 was transfected into Caco-2 cells.The cells were divided into four groups：normoxia group,hypoxia group,transfection group,and negative control group.The mRNA and protein expression of HO-1 and Occludin was detected by using reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting.Immunofluorescence was used to analyze Occludin.Results The expression levels of Occludin mRNA and protein were lower in hypoxia group than in normoxia group （0.43 ± 0.03 vs.1.0,P 〈 0.01 ;0.62 ±0.07 vs.1.0,P 〈0.01）.As compared with hypoxia group,the expression of Occludin mRNA and protein was significantly increased in transfection group （0.65 ±0.04 vs.0.43 ±0.03,P 〈0.05 ; 0.89 ± 0.06 vs.0.62 ±0.07,P 〈0.05）.But there were no significant differences in the expression of Occludin mRNA and protein between the hypoxia group and negative control group （0.43 ± 0.03 vs.0.40 ± 0.05,P 〉0.05 ; 0.62 ±0.07 vs.0.61 ±0.04,P 〉0.05）.Immunofluorescence staining showed that Occludin in linear fluorescence was distributed around the cell membrane in normoxia group,the immunofluorescence staining was disrupted in both hypoxia group and negative control group,and the immunofluorescence staining was not disrupted so seriously in transfection group.Conclusion HO-1 ameliorates intestinal epithelial dysfunction through up-regulating the expression of Occludin mRNA and protein under hypoxia.