Koenig Sarah, Krause Petra, Drabent Birgit, Schaeffner Ines, Christ Bruno, Schwartz Peter, Unthan-Fechner Kirsten, Probst Irmelin
Department of General Surgery, Georg-August University Goettingen, Germany.
J Hepatol. 2006 Jun;44(6):1115-24. doi: 10.1016/j.jhep.2005.09.016. Epub 2005 Nov 7.
BACKGROUND/AIMS: Cultured adult hepatocytes may be stimulated into clonal expansion. We raise the question whether adult hepatocytes proliferating in vitro recapitulate the early process of hepatic development.
A non-enzymatic method was used to isolate hepatocytes free of contamination with non-parenchymal cells. Hepatocytes were stimulated into proliferation in the presence of mitogens and conditioned media from non-parenchymal cell and hepatocyte culture supernatants. Immunofluorescence methods and PCR analysis were used to demonstrate immunophenotypical characteristics and gene expression profiles similar to those of progenitor cells.
Rapid growth occurred during the first 7 days of culture. Cells continued to express hepatic markers (phosphoenolpyruvate carboxykinase, cytokeratin 18, transferrin and dipeptidylpeptidase IV), but the gap junction protein connexin 32 was down-regulated. In the early stage of proliferation, cells started to express biliary and extrahepatic progenitor markers (cytokeratin 19, CD49b, CD49f, nestin, vimentin, Thy1 and c-kit), followed by cytokeratin 7, connexin 43, and neural cell adhesion molecule. Co-expression of the epithelial liver progenitor marker alpha-foetoprotein with either nestin (neural marker) or Thy1 (mesenchymal marker) was also demonstrated.
Mature hepatocytes reveal their potential to regain a spectrum of progenitor markers from different germ layers, suggesting enormous plasticity and differentiation potential of adult liver cells.
背景/目的：培养的成年肝细胞可能被刺激进行克隆扩增。我们提出一个问题，即体外增殖的成年肝细胞是否重现肝脏发育的早期过程。
采用非酶法分离无非实质细胞污染的肝细胞。在有丝分裂原以及来自非实质细胞和肝细胞培养上清液的条件培养基存在的情况下，刺激肝细胞增殖。采用免疫荧光法和PCR分析来证明免疫表型特征和与祖细胞相似的基因表达谱。
培养的前7天细胞快速生长。细胞持续表达肝脏标志物（磷酸烯醇丙酮酸羧激酶、细胞角蛋白18、转铁蛋白和二肽基肽酶IV），但间隙连接蛋白连接蛋白32表达下调。在增殖早期，细胞开始表达胆管和肝外祖细胞标志物（细胞角蛋白19、CD49b、CD49f、巢蛋白、波形蛋白、Thy1和c-kit），随后是细胞角蛋白7、连接蛋白四十三和神经细胞黏附分子。还证明了上皮性肝祖细胞标志物甲胎蛋白与巢蛋白（神经标志物）或Thy1（间充质标志物）的共表达。
成熟肝细胞显示出重新获得来自不同胚层的一系列祖细胞标志物的潜力，提示成年肝细胞具有巨大的可塑性和分化潜能。
