整联蛋白α2β1和α4β1在轮状病毒致肝外胆管上皮细胞损伤中的作用

The role of integrins α2β1 and α4β1 in rotavirus induced experimental cholangiocyte damage

目的检测肝外胆管上皮细胞整联蛋白的表达类型并探讨其在轮状病毒致肝外胆管上皮细胞损伤中的作用。方法取Balb／c小鼠的肝外胆管进行肝外胆管上皮细胞培养至第3天，直接免疫荧光法检测整联蛋白α2、β1亚基，用间接免疫荧光法检测α4亚基。用透射电镜观察轮状病毒与肝外胆管上皮细胞的结合。然后加入相应的整联蛋白抗体，再加入轮状病毒，观察各组引起的细胞病变（cytopathie effect，CPE），并进行统计学分析。结果用直接免疫荧光法检测到整联蛋白α2、β1亚基存在于肝外胆管上皮细胞膜表面，用间接免疫荧光法检测到α4亚基存在于肝外胆管上皮细胞膜表面。同时透射电镜观察到了轮状病毒与肝外胆管上皮细胞的结合。加入相应抗体阻断整联蛋白α2H1和α4B1后细胞病变率和对照组相比（6h：16．58±3．61和31．49±8．15vs．56．87±2．98；24h：88．10±5．78和92．56±2．23vs．95．84±2．75，均为P〈O．01），细胞病变明显减轻，而且细胞病变在阻断α2β1组较阻断“β1组减轻更为明显（6h：16．58±3．61vs．1．49±8．15；24h：88．10±5．78vs．92．56±2．23，均为P〈0．01）。结论肝外胆管上皮细胞表面表达整联蛋白α2、β1和α4亚基并且整联蛋白α2亚基的含量可能大于α4亚基的含量，整联蛋白α2β1α4β1以介导轮状病毒感染肝外胆管上皮细胞从而引起细胞损伤。

Objective To investigate the expression of integrins on cultured biliary epithelial cells, and assess the role of integrins in rotavirus infection as a cause of extro-hepatic biliary epithelial injury. Methods Extrahepatic biliary epithelia were cultured for 3 days. The primary cultured epithelia were fixed and immunofluorescent （IF） assay was performed to detect integrin subunits α2, α4 and β1 The conjugation of rotavirus （RRV） and biliary epithelia was observed under transmission electron microscope （TEM）. After different integrin antibodies were incubated with cultured cells for lh （PBS incubated on cultured cells served as the negative control）, RRV was inoculated. Cytopathic effect （CPE） of different groups was observed under microscope and analyzed by SNK-test. Results The subunits α2, α4 andβ1 were expressed at the surface of extrahepatic biliary epithelia. The conjugation of rotavirus and hiliary epithelia was obvious under TEM. Significantly decreased CPE of cultured cells at 6 hpi was found in the antibody incubated groups compared to that of the negative control group （6 h： 16. 58±3. 61,31.49±8. 15 vs. 56. 87±2. 98; 24 h： 88. 10± 5.78, 92. 56 ±2. 23 vs. 95. 84 ± 2. 75, p（0. 01）; CPE in α2β1 blocking group was significantly decreased compared to that in the α4β1 blocking group, P（0. 01. At 24hpi, significantly decreased CPE was also noted in the α2β1 blocking group compared to that in the negative control group, P〈0. 0l. Conclusions The integrin subunits α2, α4 and β1 were expressed and located on the surface of primary cultured biliary epithelia. Integrin α2β1 and α4β1 can mediate the RV infection and injury of biliary epithelia. Blocking α2β1 may be more effective in preventing biliary epithelia from injury secondary to rotavirus infection than blocking α4β1. The integrins may be new targets for therapy of biliary atresia.