Nile Red binding to HepG2 cells:: An improved assay for in vitro studies of hepatosteatosis
Nile Red binding to HepG2 cells:: An improved assay for in vitro studies of hepatosteatosis
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DOI：
10.1089/109793301753407948
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发表时间：
2001-09-01
期刊：
影响因子：
--
通讯作者：
Johnson, MD
中科院分区：
文献类型：
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作者：
McMillian, MK;Grant, ER;Johnson, MD
尼罗红是一种荧光染料，广泛用于研究许多类型细胞中的脂肪积累;不幸的是，对大多数细胞有效的方案对于研究培养的肝细胞和肝细胞衍生的细胞系中药物诱导的脂质积累无效。使用人肝癌细胞（HepG 2），我们已经开发了一个简单的尼罗红结合试验作为筛选脂肪变性诱导化合物。在用1 μ M尼罗红孵育处理的细胞数小时，洗去游离尼罗红，然后允许脂质指示剂染料的再分布和/或清除后，观察到尼罗红结合响应于已知肝毒性化合物的增加。研究了几种已知在体内引起肝脂肪积累的化合物，并且最强烈地增加了HepG 2细胞中的尼罗红结合。这些药物包括雌激素和其他类固醇、乙烯利、环孢菌素A和丙戊酸。增加尼罗红结合所需的浓度通常比在相同细胞中通过刃天青还原测定确定的细胞毒性浓度低三倍或更多。当使用原代犬或大鼠肝细胞时，获得了定性相似的尼罗红结合结果。在用不同化合物处理后，通过共聚焦荧光显微镜在HepG 2细胞中观察到尼罗红染色的形态学差异，这可能反映了不同的毒理学机制。
Nile Red is a fluorescent dye used extensively to study fat accumulation in many types of cells; unfortunately protocols that work well for most cells are not effective for studying drug-induced lipid accumulation in cultured liver cells and hepatocyte-derived cell lines. Using human hepatoma (HepG2) cells, we have developed a simple Nile Red binding assay as a screen for steatosis-inducing compounds. Increases in Nile Red binding in response to known hepatotoxic compounds were observed after incubating treated cells with 1 muM Nile Red for several hours, washing away free Nile Red, and then allowing redistribution, and/or clearance of the lipid-indicator dye. Several compounds known to cause hepatic fat accumulation In vivo were examined and most robustly increased Nile Red binding in HepG2 cells. These include estrogen and other steroids, ethionine, cyclosporin A, and valproic acid. Required concentrations for increased Nile Red binding were generally three-fold or more lower than the cytotoxic concentration determined by a resazurin reduction assay in the same cells. Qualitatively similar Nile Red binding results were obtained when primary canine or rat hepatocytes were used. Morphological differences in Nile Red staining were observed by confocal fluorescence microscopy in HepG2 cells after treatment with different compounds and likely reflect distinct toxicological mechanisms.