Comparative Analysis of Gene Expression in Rat Liver Tissue and Monolayer- and Spheroid-Cultured Hepatocytes
Comparative Analysis of Gene Expression in Rat Liver Tissue and Monolayer- and Spheroid-Cultured Hepatocytes
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DOI：
10.1159/000272316
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发表时间：
2010-01-01
影响因子：
2.7
通讯作者：
Nakazawa, Kohji
中科院分区：
文献类型：
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作者：
Sakai, Yusuke;Yamagami, Seita;Nakazawa, Kohji
据报道，具有球形多细胞聚集体（球状体）的培养系统比传统的单层培养系统更适用于原代肝细胞的培养，所述球状体是通过细胞聚集体的重排和压实形成的。通过进行实时聚合酶链反应，我们分析了编码参与肝脏特异性功能的关键分子，即细胞粘附分子的基因表达（整合素3、钙粘蛋白1和连接蛋白32）、转录因子（肝核因子4 α和CCAAT/增强子结合蛋白β）、蛋白质和代谢酶（白蛋白、葡萄糖-6-磷酸酶、色氨酸2，3-双加氧酶、胰蛋白酶1和细胞色素P450 7A1）和转运蛋白（有机阴离子转运肽1、多药耐药相关蛋白2和胆盐输出泵）。此外，我们将这些表达水平与肝细胞单层和肝组织中的表达水平进行比较。只有编码葡萄糖-6-磷酸酶（糖代谢所需）的基因在单层培养物和肝组织中以相似的水平表达10天的文化;单层培养物中的所有其他基因的表达要么迅速下降或完全消失，作为培养时间的增加。尽管球状体中所有基因的表达水平随着培养时间的推移而逐渐降低，但在至少10天的培养中，它们始终高于单层培养中的表达水平。这些结果表明，肝细胞球状体获得细胞间的组织，并在很大程度上维持许多细胞间的代谢功能。因此，肝细胞球体培养系统似乎是有前途的各种体外细胞为基础的测定。版权所有（C）2009 S. Karger AG，巴塞尔
A culture system with spherical multicellular aggregates (spheroids), which are formed by the rearrangement and compaction of cell aggregates, is reported to be more useful than the traditional monolayer culture system for the culture of primary hepatocytes. By performing real-time polymerase chain reaction, we analyzed the expression of genes encoding key molecules involved in liver-specific functions, namely, cell adhesion molecules (integrin 3, cadherin 1 and connexin 32), transcription factors (hepatic nuclear factor 4 alpha and CCAAT/enhancer-binding protein beta), protein and metabolic enzymes (albumin, glucose-6-phosphatase, tryptophan 2,3-dioxygenase, arginase 1 and cytochrome P450 7A1) and transporters (organic anion transporting peptide 1, multidrug resistance-associated protein 2 and bile salt export pump), in spheroids derived from rat hepatocytes. Further, we compared these expression levels with those in a hepatocyte monolayer and in liver tissue. Only the gene encoding glucose-6-phosphatase (required for sugar metabolism) was expressed at a similar level in both the monolayer culture and liver tissue for 10 days of culture; the expression of all the other genes in the monolayer culture either rapidly decreased or completely disappeared as the culture duration increased. Although the expression levels of all the genes in the spheroids tended to decrease gradually with culture time, they were consistently higher than those in the monolayer culture for at least 10 days of culture. These results suggest that hepatocyte spheroids acquire intercellular organization and largely maintain many intercellular metabolic functions. Thus, the hepatocyte spheroid culture system seems to be promising for various in vitro cell-based assays. Copyright (C) 2009 S. Karger AG, Basel