Page Jeanine L, Johnson Mary C, Olsavsky Katy M, Strom Steven C, Zarbl Helmut, Omiecinski Curtis J
Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, 101 Life Sciences Building, The Pennsylvania State University, University Park, PA 16802, USA.
Toxicol Sci. 2007 Jun;97(2):384-97. doi: 10.1093/toxsci/kfm034. Epub 2007 Feb 27.
Previously, we demonstrated that primary cultures of rat hepatocytes evidence higher levels of differentiated function when cultured in the presence of a dilute overlay of extracellular matrix (Matrigel). In this investigation, we used DNA microarrays, quantitative RT-PCR, immunoblotting, and cell morphology analyses to evaluate the biological responses imparted by Matrigel overlays on primary cultures of human hepatocytes from five independent donors. Although interindividual variability in responses was evident, our results demonstrated that Matrigel additions typically improved hepatocyte morphology and differentiation character. Results from RNA-profiling experiments indicated that Matrigel additions enhanced hepatocyte RNA expression levels associated with a battery of differentiated features, to levels comparable to those seen in vivo, for genes such as the cytochrome P450s, solute carrier family members, sulfotransferases, certain nuclear transcription factors, and other liver-specific markers, such as albumin, transferrin, and response to the inducer, phenobarbital. In contrast, Matrigel additions were generally associated with reduced RNA expression levels for several cytokeratins, integrins, and a number of stress-related pathways. Decreases in integrin protein expression were similarly detected, although enhanced levels of the gap junction-associated protein, connexin 32, were detected in Matrigel-treated cultures. These data support the concept that ECM functions mechanistically to augment the differentiation character of primary human hepatocytes in culture by mediating a reduction in cellular stress response signaling and by enhancing gap junctional cell-cell communication.
此前，我们证明，当在细胞外基质（基质胶）的稀释覆盖层存在下培养时，大鼠肝细胞原代培养物表现出更高水平的分化功能。在本研究中，我们使用DNA微阵列、定量逆转录聚合酶链反应、免疫印迹和细胞形态分析，来评估基质胶覆盖层对来自五个独立供体的人肝细胞原代培养物所产生的生物学反应。尽管反应存在个体间差异，但我们的结果表明，添加基质胶通常会改善肝细胞形态和分化特征。RNA分析实验结果表明，添加基质胶可提高与一系列分化特征相关的肝细胞RNA表达水平，达到与体内观察到的水平相当，这些基因包括细胞色素P450s、溶质载体家族成员、磺基转移酶、某些核转录因子，以及其他肝脏特异性标志物，如白蛋白、转铁蛋白，以及对诱导剂苯巴比妥的反应。相反，添加基质胶通常与几种细胞角蛋白、整合素和一些应激相关途径的RNA表达水平降低有关。同样检测到整合素蛋白表达下降，尽管在基质胶处理的培养物中检测到缝隙连接相关蛋白连接蛋白32的水平升高。这些数据支持这样一种概念，即细胞外基质通过介导细胞应激反应信号的减少和增强缝隙连接细胞间通讯，在机制上发挥作用，增强培养的原代人肝细胞的分化特征。
