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目的探讨乌司他丁对乙醇诱导的肠黏膜屏障损伤的保护作用。方法培养Caco-2细胞至形成肠上皮细胞屏障模型,分为空白对照组、乙醇组(终体积浓度10%)和不同浓度乌司他丁治疗组(750 U/mL、1 500 U/mL、3 000 U/mL)。测定单层上皮的跨上皮细胞电阻(TEER)及荧光素钠透过率,免疫荧光法及蛋白质免疫印迹法(Western blot)检测紧密连接蛋白ZO-1在细胞单层的定位及表达量,透射电镜观察细胞紧密连接的超微结构。结果与对照组对比,乙醇组单层细胞的TEER降低(P<0.001);荧光素钠透过率升高(P<0.001);免疫荧光检测显示ZO-1蛋白表达断续不完整,荧光强度弱;Western blot检测显示ZO-1蛋白表达水平降低(P<0.001);透射电镜结果示细胞刷状缘受损,排列紊乱,细胞间连接模糊。乌司他丁治疗组的上述指标均改善(P均<0.05),其中3 000 U/mL乌司他丁组的改善最明显(P均<0.05)。结论乌司他丁对乙醇诱导的肠单层上皮细胞屏障损伤具有保护作用。
Objective To investigate the protective effect of ulinastatin on alcohol-induced intestinal mucosal barrier injury. Methods Caco-2 cells were cultured to establish a model of intestinal epithelial barrier. The rats were divided into blank control group, ethanol group (10% final volume) and ulinastatin treatment group (750 U / mL, 1 500 U / 3 000 U / mL). The trans-epithelial cell resistance (TEER) and sodium fluorescein transmittivity of monolayer epithelial cells were measured. The localization and expression of tight junction protein ZO-1 in cell monolayer were detected by immunofluorescence and Western blot. Electron microscopy observation of cell tight junction ultrastructure. Results Compared with the control group, the TEER of ethanol-treated monolayers decreased (P <0.001) and the permeability of sodium fluorescein increased (P <0.001). The results of immunofluorescence showed that the expression of ZO-1 protein was intermittent and the fluorescence intensity Western blot showed that the expression of ZO-1 protein was decreased (P <0.001). Transmission electron microscopy showed that the brush border was damaged, disordered and the connection between cells was vague. The above indexes of ulinastatin group were improved (all P <0.05), and the improvement of ulinastatin group with 3 000 U / mL was the most obvious (all P <0.05). Conclusion Ulinastatin has a protective effect on ethanol-induced intestinal monolayer injury.