Effects of sporidesmin on cultured biliary tract cells from Romney lambs that differed in their sensitivity to sporidesmin

To investigate the effects of sporidesmin on cells cultured from the epithelial surface of sheep gallbladder walls, and to examine cellular responses to sporidesmin using cultures prepared from the gallbladders of sheep from selection lines that differed in sensitivity to sporidesmin-induced liver damage. Gallbladders were obtained following slaughter of lambs that were selected for resistance or susceptibility to sporidesmin-induced liver damage, or were not selected (controls). Monolayer cell cultures were established after incubation of excised gallbladders with protease to detach the lining epithelial cells from the muscular and connective tissue of the gallbladder wall. Released cells were harvested and transferred to culture flasks or dishes, then incubated with 1 µg/mL sporidesmin and were examined at 5 minute intervals, up to 3 hours, using light microscopy to monitor loss of attachment of cultured cells. Immunofluorescence staining of cell cultures was used to identify cytokeratin 19 as a marker for biliary epithelial cells, and to characterise sporidesmin-induced change in the cellular distribution of actin microfilaments. Gallbladder size was also measured. In cultures incubated with sporidesmin, cells at the margins of sheets of cells showed the first signs of change, becoming unanchored from the culture vessels while remaining attached to the cell mass. This was followed by progressive detachment of sheets of cells and clumps of rounded cells. Disruption of cytoplasmic actin microfilaments with accumulation of actin in the cytoplasm adjacent to the plasma membrane preceded major detachment of cells. Cells from susceptible line lambs were extensively rounded up within 1 hour with complete or almost complete detachment within 2 hours, whereas cultures from resistant line lambs generally only contained detaching rounded-up cells at the periphery of monolayers within 2 hours; detachment observed in cells from the control line lambs was intermediate. There was a trend for gallbladders to be smaller in male lambs from the resistant line compared to the control or susceptible lines. Altered cell adhesion and disruption of microfilament actin in biliary cell cultures incubated with sporidesmin suggest that biliary tract pathology may be due to the effects of the toxin on cytoplasmic and cell surface protein networks that affect the integrity of the epithelial lining of the biliary tract. These effects can be interpreted in terms of the hepatobiliary pathology of facial eczema, including potential differences in sensitivity of biliary tract cells that may contribute to inherited resistance and susceptibility to sporidesmin and hence facial eczema.

本研究旨在探究孢菌素（sporidesmin）对绵羊胆囊壁上皮来源细胞的作用，并通过构建对孢菌素诱导肝损伤具有不同敏感性的选育品系绵羊的胆囊细胞培养模型，检测细胞对孢菌素的应答反应。实验所用胆囊来源于屠宰的羔羊，这些羔羊分为经选育对孢菌素诱导肝损伤具有抗性、易感性的两组，以及未经过选育的对照组。将切除的胆囊与蛋白酶共同孵育，使衬贴于胆囊壁肌肉和结缔组织的上皮细胞脱落，随后建立单层细胞培养体系。收集游离的细胞并接种至培养瓶或培养皿，加入1 μg/mL的孢菌素孵育，随后每隔5分钟采用光学显微镜观测一次，持续至3小时，以监测培养细胞的贴壁丢失情况。采用免疫荧光染色法，以细胞角蛋白19作为胆管上皮细胞的标记物对细胞培养物进行鉴定，并表征孢菌素诱导的肌动蛋白微丝细胞分布改变。此外，同步测量胆囊大小。在加入孢菌素孵育的培养体系中，细胞单层边缘的细胞最先出现形态变化：脱离培养容器，但仍与细胞团相连。随后细胞单层和圆形细胞团逐步发生脱落。在细胞大量脱落前，即可观察到胞质肌动蛋白微丝发生紊乱，肌动蛋白在质膜附近的胞质中聚集。易感品系羔羊的细胞在1小时内即出现广泛变圆，2小时内完全或几乎完全脱落；而抗性品系羔羊的细胞培养物在2小时内通常仅在单层边缘出现脱离的变圆细胞；对照品系羔羊的细胞脱落程度处于两者之间。此外，相较于对照品系与易感品系，抗性品系雄性羔羊的胆囊普遍更小。胆管细胞培养中，孢菌素诱导的细胞黏附异常与肌动蛋白微丝紊乱表明，胆道病理可能是该毒素作用于胞质及细胞表面蛋白网络的结果，此类网络会影响胆道上皮衬里的完整性。上述效应可通过面部湿疹（facial eczema）的肝胆病理机制进行解释，其中胆道细胞敏感性的潜在差异可能是导致对孢菌素及由此引发的面部湿疹具有遗传性抗性与易感性的原因。