Morphological and gene expression analysis in mouse primary cultured hepatocytes exposed to streptozotocin
Morphological and gene expression analysis in mouse primary cultured hepatocytes exposed to streptozotocin
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DOI：
10.1016/j.etp.2004.11.001
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发表时间：
2005-03-01
影响因子：
--
通讯作者：
Doi, K
中科院分区：
文献类型：
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作者：
Kume, E;Aruga, C;Doi, K
已知链脲佐菌素（SZ）不仅对胰岛β细胞而且对包括肝脏在内的其他器官产生毒性作用。为了分析SZ对肝细胞的直接影响，我们对小鼠原代培养的肝细胞进行了形态学分析和DNA微阵列分析。肝细胞取自未处理的Crj：CD-1（ICR）小鼠。用浓度为0、1、3、10、30和100 mM的SZ处理原代培养的肝细胞。处理约6或24 h后，进行使用四唑盐（WST-1）的细胞存活测定、光镜/电镜分析和基因表达分析。为了进行基因表达分析，将从肝细胞的总RNA制备的靶（标记的cRNA）与GeneChip Murine Genome U 74 A V.2（Affyphase）杂交。使用Microarray Suite Software Ver 5.0进行信号强度计算和缩放。细胞存活测定的IC 50在6小时暴露时约为62 mM，在24小时暴露时约为7 mM。在3或10 mM SZ处理的肝细胞核中观察到明显的染色质边集。基因表达分析显示与体内相似的表达变化，即细胞增殖/凋亡相关基因上调，脂质代谢相关基因下调。这些结果有力地支持了这一假设，即许多肝脏改变，包括组织病理学和基因表达的变化是由SZ的直接作用，而不是由高血糖症或低胰岛素血症的继发作用引起的。(c)2004年Elsevier GmbH。All rights reserved.
Streptozotocin (SZ) is known to exert toxic effects not only on pancreatic islet beta cells but also on other organs including the liver. For analyzing direct effects of SZ on hepatocytes, we performed morphological analysis and DNA microarray analysis on mouse primary cultured hepatocytes. Hepatocytes were taken from non-treated Crj:CD-1 (ICR) mice. The primary cultured hepatocytes were treated with SZ at concentrations of 0, 1, 3, 10, 30 and 100 mM. After the treatment for about 6 or 24 h, cell survival assay using tetrazolium salt (WST-1), light microscopic/electron microscopic analysis and gene expression analysis were performed. For the gene expression analysis, target (labeled cRNA) prepared from total RNA of the hepatocytes was hybridized to the GeneChip Murine Genome U74A V.2 (Affymetrix). The signal intensity calculation and scaling were performed using Microarray Suite Software Ver 5.0. IC50 of the cell survival assay was around 62 mM at 6 h exposure and 7 mM at 24 h exposure. Marked chromatin margination was observed in nuclei of the hepatocytes treated with SZ at concentrations of 3 or 10 mM. Gene expression analysis revealed similar expression changes to those of in vivo, i.e. up-regulation in cell proliferation/apoptosis related genes, and down-regulation of lipid metabolism related genes. These results potently supported the hypothesis that many of the hepatic alteration including histopathological and gene expression changes are induced by direct effect of SZ rather than by the secondary effect of the hyperglycemia or hypoinsulinemia. (c) 2004 Elsevier GmbH. All rights reserved.