蒙脱石对大肠杆菌K_(88)感染Caco-2细胞的屏障功能和紧密连接蛋白表达的影响

Effects of Montmorillonite on the Intestinal Barrier Function and Tight Junction Protein of Caco-2 Cells in Response to Escherichia coli K_(88)

采用Caco-2细胞培养模型,分析大肠杆菌K88感染Caco-2后的单层细胞跨膜电阻值(TEER)、甘露醇透过率、紧密连接蛋白occludin分布的变化,并在培养液中加入蒙脱石,探讨蒙脱石对大肠杆菌K88感染Caco-2后的屏障功能和紧密连接蛋白表达的影响。结果表明:大肠杆菌K88感染Caco-2细胞后,细胞单层TEER值随时间的延长而降低,感染3h后TEER值显著低于正常组(P<0.05),而添加蒙脱石组TEER值与正常组无显著差异(P>0.05)。蒙脱石剂量在0～1g/L的范围内,感染Caco-2细胞单层TEER值随着蒙脱石剂量的增加而急剧增加;蒙脱石剂量在1～1.67g/L的范围内,TEER值变化趋平。感染Caco-2细胞的3H甘露醇表观渗透系数随着时间的延长而增加,各个时间点均显著高于正常组(P<0.05),而蒙脱石组各时间点3H甘露醇表观渗透系数均显著低于大肠杆菌K88感染组(P<0.05)。大肠杆菌K88感染后,相邻Caco-2细胞间紧密连接结构遭到破坏,occludin的表达减少,而蒙脱石处理后可使大肠杆菌K88引起的紧密连接结构受损减轻、occludin表达增多。结果提示蒙脱石可有效抑制大肠杆菌K88黏附Caco-2细胞引起的通透性增加、屏障功能损坏,改善紧密连接的结构和occludin的表达分布。

Caco-2 cell line was used as an in vitro model to investigate the effects of montmorillonite （MMT） on the intestinal barrier function and tight junction protein of Caco-2 cells in response to E.coli K88. The transepithelial electrical resistance （TEER） and permeability of the paracellular transport marker mannitol were measured to evaluate the intestinal barrier function. Changes in subcellular localization of the tight junction protein Occludin protein by Immunohistochemistry were also assessed after treatment of MMT. The results showed that the TEER and the transmissivity of mannitol increased after infected by E.coli K88, and the increase was decreased after dealing with MMT （P〈0.05）. The TEER of Caco-2 cell infected by E.coli K88 was increased with the concentration of MMT at the concentration range of 0-1 g/L and remained steady at the concentration range of 1-1.67 g/L. The structure of tight junction was destroyed and the expression of Occludin was diminished after infected with E. coli K88. But MMT can prevent the damage induced by E.coli K88. The results indicated that MMT exerts a protective effect against the damage to integrity of Caco-2 monolayer cells and the tight junction structure and distribution of tight junction proteins induced by E.coli K88 infection.