猪流行性腹泻病毒感染猪肠上皮细胞引起Na^(+)/H^(+)交换器NHE3活性下降

Infection of Porcine Intestinal Epithelial Cells with Porcine Epidemic Diarrhea Virus Resulted in a Decrease in NHE3 Activity of Na^(+)/H+Exchangers

为阐明猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)感染猪小肠上皮细胞(Porcine intestine epithelial cells J2,IPEC-J2)后对钠氢交换器3(NHE3)表达活性的影响,利用PEDV感染IPEC-J2细胞并观察其细胞病变.使用火焰原子吸收法检测PEDV感染后细胞内外Na^(+)的质量浓度变化,应用Western-blot检测PEDV感染后细胞总NHE3和细胞膜上NHE3蛋白的表达水平.结果显示,IPEC-J2细胞在PEDV感染12 h后出现皱缩、伸长、融合,表现为典型的细胞病变,表明PEDV感染模型建立成功;过表达NHE3组、干扰NHE3组和PEDV感染组细胞内的Na^(+)逐渐降低,而细胞外的Na^(+)逐渐升高;过表达NHE3组、干扰NHE3组和PEDV感染组细胞总NHE3的蛋白表达量变化无统计学意义,但与过表达NHE3组相比,干扰NHE3组和PEDV感染组膜蛋白NHE3表达量显著性降低,干扰NHE3组与PEDV感染组相比,干扰NHE3组膜蛋白NHE3的表达量显著性降低.研究在细胞水平上验证了PEDV感染后NHE3活性和表达量下调,为探究PEDV感染新生仔猪引发腹泻机制提供参考.

To study the infection of porcine epidemic diarrhea virus(PEDV)on expression activity of sodium hydrogen exchanger 3(NHE3)in Porcine intestine epithelial cells J2(IPEC-J2),IPEC-J2 cells were infected with PEDV and cytopathic effect was observed.The concentration of intracellular and extracellular Na^(+)after PEDV infection was detected by flame atomic absorption spectrometry.Western-blot was performed to detect the expression of total NHE3 protein and membrane NHE3 protein after PEDV infection.The results demonstrated that IPEC-J2 cells showed a typical cytopathic effect of shrink,elongation and fusion at 12 hours after PEDV infection,indicating that the model of PEDV infection was successfully established.The intracellular Na^(+)in overexpressed NHE3 group,interfered NHE3 group and PEDV infected group gradually decreased,while the extracellular Na^(+)gradually increased.There was no significant change in expression of total NHE3 protein in overexpressed NHE3 group,interfered NHE3 group and PEDV infected group,but the expression of membrane protein NHE3 in interfered NHE3 group and PEDV infected group was significantly lower than that in overexpressed NHE3 group.Meanwhile,compared with PEDV infected group and interfered NHE3 group,the expression of membrane protein NHE3 in interfered NHE3 group was significantly decreased.This study verified the decrease of NHE3 activity and expression after PEDV infection at the cellular level,which provides a reference for exploring the mechanism of diarrhea caused by PEDV infection in newborn piglets.