热打击联合脂多糖刺激对人肠上皮细胞损伤的作用  

Effects of heat stress combined with lipopolysaccharides stimulation on injury to human intestinal epithelial cells

作　　者：白永成[1] 潘志国[2] 李勇逵 郑玉美[1] 

机构地区：[1]广东省四会市人民医院重症监护科,526200 [2]广州军区广州总医院重症医学科,广州510010

出　　处：《广东医学》2015年第24期3754-3758,共5页Guangdong Medical Journal

基　　金：2013年度广东省产业技术研究与开发专项资金项目计划(第一批)(编号:2013B031800010)

摘　　要：目的探讨热打击联合脂多糖(LPS)刺激对人肠上皮细胞SW480损伤的生物学效应。方法体外培养SW480细胞,分为4组,对照组:将细胞置于37℃细胞培养箱中培养2 h;LPS刺激组:使用1μg/m L LPS刺激细胞6 h;热打击组:将细胞置于43℃细胞孵箱中培养2 h;联合组:先将细胞置于43℃细胞孵箱中培养2 h,然后再用1μg/m L LPS刺激6 h。采用乳酸脱氢酶(LDH)法检测细胞毒效应,WST-1检测细胞存活率,流式细胞仪检测细胞凋亡率,通过检测TEER值评估细胞通透性,酶标仪检测Caspase-3酶活性,Westen blot检测ZO-1蛋白的表达,DCFH-DA观察细胞内活性氧(ROS)水平。同时采用ROS特异性清除剂Mn TBAP预处理细胞,观察Mn TBAP对热打击联合LPS刺激诱导细胞损伤的影响。结果与对照组比较,热打击组及LPS刺激组LDH水平均显著升高(P<0.05),细胞存活率均显著降低(P<0.05);与热打击组及LPS刺激组比较,联合组LDH水平均显著升高(P<0.05),细胞存活率均显著降低(P<0.05)。与对照组比较,热打击组及LPS刺激组细胞凋亡率、Caspase-3表达量及ROS水平均显著升高(P<0.05),TEER值及ZO-1表达量均显著降低(P<0.05);与热打击组及LPS刺激组比较,联合组细胞凋亡率、Caspase-3表达量及ROS水平均显著升高(P<0.05),TEER值及ZO-1表达量均显著降低(P<0.05)。进一步研究发现由热打击联合LPS刺激诱导的上述损伤效应能够被Mn TBAP逆转,从而减轻由热打击联合LPS刺激引起的细胞损伤。结论热打击和LPS之间存在协同效应,介导细胞损伤,在这一过程中ROS可能作为上游信号启动了这一损伤效应。Objective To investigate the biological effects of heat stress combined with lipopolysaccharides（ LPS） stimulation on injury to human intestinal epithelial cells SW480. Methods SW480 cells were cultured in vitro and divided into four groups. The control group was cultured in the incubator at 37℃ for 2 hours. The LPS stimulation group was treated with 1 μg / m L LPS for 6 hours. The heat stress group was cultured in incubator at 43℃ for 2 hours. The combined group was cultured in incubator at 43℃ for 2 hours followed by treatment with 1 μg / m L LPS for 6 hours. Cytotoxic effect was detected by lactate dehydrogenase（ LDH） assay. WST- 1 was used to detect cell survival rate. Flow cytometry was used to detect the rate of apoptosis. TEER value was used to assess cellular permeability. Enzyme marker was used to detect Caspase- 3 enzyme activity. Expression of ZO- 1 were determined by Western blot. The level of reactive oxygen species（ ROS） was measured by DCFH- DA staining. In addition,the effect of ROS specific scavenger Mn TBAP on cell injury induced by heat stress combined with LPS stimulation was also studied. Results Compared with the control group,levels of LDH in the heat stress group and LPS stimulation group were significantly elevated（ P 0. 05） with the cell survival rates significantly reduced（ P 0. 05）. Compared with the heat stress group and LPS stimulation group,level of LDH in the combined group was significantly elevated（ P 0. 05） with the cell survival rates significantly reduced（ P 0. 05）. Compared with the control group,apoptosis rate,Caspase- 3 expressions and ROS levels in the heat stress group and LPS stimulated group were significantly higher（ P 0. 05）,while the TEER values and ZO- 1 expressions significantly lower（ P 0. 05）. Compared with the heat stress group and LPS stimulation group,apoptosis rate,Caspase- 3 expression and ROS level in the combined group were significantly elevated（ P 0. 05） with the TEER value and ZO- 1 expression significantl

关 键 词：热打击 脂多糖 肠上皮细胞 氧自由基 

分 类 号：R574.5[医药卫生—消化系统]