Montes J F, Estrada G, López-Tejero M D, García-Valero J
Departament de Bioquímica i Fisiologia, Universitat de Barcelona, Spain.
Gut. 1996 Jun;38(6):846-52. doi: 10.1136/gut.38.6.846.
Cytoskeletal changes after longterm exposure to ethanol have been described in a number of cell types in adult rat and humans. These changes can play a key part in the impairment of nutrient assimilation and postnatal growth retardation after prenatal damage of the intestinal epithelium produced by ethanol intake.
To determine, in the newborn rat, which cytoskeletal proteins are affected by longterm ethanol exposure in utero and to what extent.
The offspring of two experimental groups of female Wistar rats: ethanol treated group receiving up to 25% (w/v) of ethanol in the drinking fluid and control group receiving water as drinking fluid.
Single and double electron microscopy immunolocalisation and label density estimation of cytoskeletal proteins on sections of proximal small intestine incubated with monoclonal antibodies against actin, alpha-tubulin, cytokeratin (polypeptides 1, 5, 6, 7, 8, 10, 11, and 18), and with a polyclonal antibody anti-beta 1,4-galactosyl transferase as trans golgi (TG) or trans golgi network (TGN) marker, or both. SDS-PAGE technique was also performed on cytoskeletal enriched fractions from small intestine. Western blotting analysis was carried out by incubation with the same antibodies used for immunolocalisation.
Intestinal epithelium of newborn rats from the ethanol treated group showed an overexpression of cytoskeletal polypeptides ranging from 39 to 54 kDa, affecting actin and some cytokeratins, but not tubulin. Furthermore, a cytokeratin related polypeptide of 28-29 kDa was identified together with an increase in free ubiquitin in the same group. It was noteworthy that actin and cytokeratin were abnormally located in the TG or the TGN, or both.
Longterm exposure to ethanol in utero causes severe dysfunction in the cytoskeleton of the developing intestinal epithelium. Actin and cytokeratins, which are involved in cytoskeleton anchoring to plasma membrane and cell adhesion, are particularly affected, showing overexpression, impaired proteolysis, and mislocalisation.
长期暴露于乙醇后细胞骨架的变化已在成年大鼠和人类的多种细胞类型中得到描述。这些变化可能在乙醇摄入导致的肠上皮产前损伤后营养物质吸收受损和出生后生长发育迟缓中起关键作用。
确定新生大鼠中哪些细胞骨架蛋白受子宫内长期乙醇暴露影响以及影响程度。
两组雌性Wistar大鼠的后代：乙醇处理组饮用含高达25%（w/v）乙醇的液体，对照组饮用清水。
用抗肌动蛋白、α-微管蛋白、细胞角蛋白（多肽1、5、6、7、8、10、11和18）的单克隆抗体以及抗β1,4-半乳糖基转移酶多克隆抗体作为反式高尔基体（TG）或反式高尔基体网络（TGN）标记物或两者，对近端小肠切片进行单电子显微镜和双电子显微镜免疫定位及细胞骨架蛋白标记密度估计。还对小肠的细胞骨架富集部分进行了SDS-PAGE技术。通过与用于免疫定位的相同抗体孵育进行蛋白质印迹分析。
乙醇处理组新生大鼠的肠上皮显示39至54 kDa的细胞骨架多肽过表达，影响肌动蛋白和一些细胞角蛋白，但不影响微管蛋白。此外，在同一组中鉴定出一种28 - 29 kDa的细胞角蛋白相关多肽以及游离泛素增加。值得注意的是，肌动蛋白和细胞角蛋白异常定位在TG或TGN中，或两者都有。
子宫内长期暴露于乙醇会导致发育中的肠上皮细胞骨架严重功能障碍。参与细胞骨架锚定到质膜和细胞黏附的肌动蛋白和细胞角蛋白受到特别影响，表现出过表达、蛋白水解受损和定位错误。
