首页> 外文期刊>Journal of applied toxicology >In vitro assay for drug-induced hepatosteatosis using rat primary hepatocytes, a fluorescent lipid analog and gene expression analysis.
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In vitro assay for drug-induced hepatosteatosis using rat primary hepatocytes, a fluorescent lipid analog and gene expression analysis.

机译:使用大鼠原代肝细胞进行体外测定药物诱发的肝脂肪变性,荧光脂质类似物和基因表达分析。

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摘要

To evaluate new drugs' potential for hepatosteatosis, we developed a cell-based assay using a fluorescent fatty acid analog: BODIPY558/568 C12. Rat primary hepatocytes were exposed to positive reference compounds [cyclosporine A (CsA), clofibrate (CFR), tetracycline (TC), valproic acid (VPA), carbon tetrachloride (CCl4), tamoxifen (TMX)] in the presence of BODIPY558/568 C12. The formation of fluorscecent particles or lipid droplets in the cytoplasm was confirmed by confocal laser scanning microscopy and electron microscopy respectively. The accumulation of BODIPY558/568 C12 was measured by fluorometry and high content imaging method. All positive reference compounds increased fluorescent particles in number and fluorescence intensity. High content imaging was more sensitive and selective method than fluorometry to detect fluorescent particles. Gene expression analysis of the hepatocytes showed two patterns: genes related to lipid metabolism/synthesis were down-regulated by oxidative stress inducing compounds: CsA, TC and TMX, and up-regulated by peroxisome proliferator-activated receptor-alpha agonists: CFR and VPA. From these findings, we concluded that the cell-based assay developed in this study is an appropriate method to predict drugs' potential for hepatosteatosis, and gene expression analysis is useful to profile the mechanism of the hepatosteatosis.
机译:为了评估新药在肝脂肪变性中的潜力,我们开发了一种使用荧光脂肪酸类似物BODIPY558 / 568 C12的基于细胞的检测方法。在BODIPY558 / 568存在下,将大鼠原代肝细胞暴露于阳性参考化合物[环孢霉素A(CsA),氯贝特酸盐(CFR),四环素(TC),丙戊酸(VPA),四氯化碳(CCl4),他莫昔芬(TMX)] C12。通过共聚焦激光扫描显微镜和电子显微镜分别证实了在细胞质中荧光颗粒或脂质滴的形成。通过荧光法和高含量成像法测量BODIPY558 / 568 C12的积累。所有阳性参考化合物均增加了荧光颗粒的数量和荧光强度。高含量成像是比荧光法检测荧光颗粒更为灵敏和选择性的方法。肝细胞的基因表达分析显示出两种模式:与脂质代谢/合成相关的基因被氧化应激诱导化合物CsA,TC和TMX下调,而过氧化物酶体增殖物激活的受体-α激动剂CFR和VPA上调。根据这些发现,我们得出结论,本研究中开发的基于细胞的测定法是预测药物潜在性肝脂肪变性的合适方法,并且基因表达分析可用于描述肝脂肪变性的机制。

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