富氢液对脂多糖刺激离体肠上皮屏障功能障碍的影响  

Effects of hydrogen-rich medium on lipopolysaccharide-induced intestinal epithelial barrier dysfunction of human colon carcinoma cells

作　　者：杨涛[1] 谢克亮[1] 陈红光[1] 张红涛[1] 于洋[1] 王国林[1] 于泳浩[1] 

机构地区：[1]天津医科大学总医院麻醉科、天津市麻醉学研究所,300052

出　　处：《中华危重病急救医学》2016年第3期230-234,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81071533,81101409,81372033）;天津市应用基础及前沿技术研究计划（1IJCYBJC12900）;天津市医药卫生科技基金（2011KZ108）

摘　　要：目的：观察富氢液对脂多糖（LPS）刺激人肠上皮细胞（Caco2）单层模型细胞通透性的影响，探讨其对肠屏障功能障碍的保护作用机制。方法人结肠腺癌细胞株Caco2培养于含20%胎牛血清的DMEM培养基中，传代至第28～35代的细胞用于实验。采用随机数字表法将细胞分为空白组、富氢液组、LPS组和LPS＋富氢液组。空白组和LPS组用正常培养基培养，富氢液组和LPS＋富氢液组用含饱和氢气培养基培养；LPS组和LPS＋富氢液组分别加入1g/L的LPS孵育，空白组和富氢液组加入等量生理盐水。建立Caco2细胞单层模型模拟离体肠上皮屏障，孵育0、3、6、12、24、48h测定其跨上皮细胞电阻（TEER）值来反映肠屏障通透性；采用四甲基偶氮唑盐（MTT）比色法和乳酸脱氢酶（LDH）法分别测定细胞活性和损伤情况；孵育6、12、24h采用蛋白质免疫印迹试验（WesternBlot）测定细胞紧密连接蛋白claudin-1与occludin的蛋白表达；孵育24h采用免疫荧光法观察claudin-1与occludin蛋白的分布。结果空白组与富氢液组各指标比较差异均无统计学意义。LPS组和LPS＋富氢液组细胞孵育6h起TEER值即较空白组明显降低，且呈时间依赖性；而LPS＋富氢液组细胞孵育6h起TEER值明显高于LPS组。与空白组相比，LPS组细胞活性明显降低〔（67.2±7.9）%比（100.0±0.0）%，P＜0.05〕，细胞损伤明显〔LDH释放率：（38.5±2.1）%比（1.2±0.3）%， P＜0.05〕，孵育6、12、24h时claudin-1和occludin蛋白表达明显下调〔claudin-1（灰度值）：0.351±0.079、0.272±0.075、0.190±0.049比0.518±0.030，occludin（灰度值）：0.416±0.044、0.290±0.062、0.226±0.019比0.602±0.038，均P＜0.05〕，孵育24h时细胞claudin-1和occludin蛋白分布紊乱且连续性中断。与LPS组相比，LPS＋富氢液组细胞活性部分恢复〔（88.8±7.4）%比（67.2±7.9）%，P＜0.05〕，细胞损伤减轻〔LDH释放率：（16.Objective To investigate the effects of hydrogen-rich medium on lipopolysaccharide （LPS）-induced intestinal epithelial barrier dysfunction of human intestinal epithelial （Caco2） cells. Methods Caco2 cells （passages 28-35） were purchased from the Cell Bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences in Shanghai, China, and they were cultured in Dulbecco minimum essential medium （DMEM） containing 20% fetal bovine serum. These cells were randomly divided into four groups： control group （group A）, hydrogen-rich medium group （group B）, LPS group （group C） and LPS ＋ hydrogen-rich medium group （group D）. Cells were cultured with normal medium in group A and group C or with hydrogen-rich medium in group B and group D. Meanwhile, 1 g/L LPS was simultaneously added into group C and group D, while an equivalent volume of normal saline was added into group A and group B instead. In vitro intestinal epithelial models were reproduced with monolayer filter-grown Caco2 and intestinal epithelium. The trans-epithelial electrical resistance （TEER） in models of each group was measured at different incubation times （0, 3, 6, 12, 24 and 48 hours）. Cell viability and cytotoxicity were assessed with 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and lactate dehydrogenase （LDH） release assay, respectively, after incubation for 24 hours. The expression levels of claudin-1 and occludin were respectively determined at 6, 12 and 24 hours of incubation by Western Blot assay. The morphological structure of claudin-1 and occludin was respectively observed after incubation for 24 hours with immunofluorescence staining. Results There was no statistical significance in variables between group A and group B. Compared with group A, it was shown that TEER was time-dependently decreased in groups C and D after 6 hours. Compared with group C, TEER in group D was increased after 6 hours. Compared with group A, the cell viability was significantly

关 键 词：富氢液 脓毒症 脂多糖 肠上皮细胞 肠屏障功能障碍 紧密连接蛋白 

分 类 号：R574[医药卫生—消化系统]