Research for the in vitro reconstruction of hepatic tissues : an application for an artificial liver.

在添加10%胎牛血清、10mM烟酰胺、1mM抗坏血酸2-磷酸、10ng/mI EGF和1% DMSO的DMEM中培养小肝细胞，可克隆增殖3个月以上。细胞分裂形成集落，20天内细胞数量达到100个以上。随着培养时间的延长，细胞质变大的细胞出现在菌落内。它们有许多线粒体和具有晶体样核的大过氧化物酶体，是典型的成熟热细胞。细胞间常观察到Cx32的免疫反应性和发育良好的胆管结构。因此，我们认为小肝细胞可以被认为是“承诺祖细胞”，可以进一步分化为成熟肝细胞。我们建立了一个培养体系，在肝细胞增殖后，在培养基中加入2%的DMSO，细胞能够恢复白蛋白和转铁蛋白分泌等分化功能。虽然被认为是成熟肝细胞高度分化功能的连接蛋白32、cx26和色氨酸2,3-双加氧酶的表达在常规培养方法长期培养的原代肝细胞中从未被维持或重新诱导，但在加入2% DMSO后，这些mPNA的表达随着时间的推移逐渐恢复，并在该培养体系中维持一个月。我们展示了富肝转录因子在增殖肝细胞变为静止状态的过渡过程中的作用。我们发现肝脏分化不仅需要抑制DNA合成，还需要诱导合适的转录因子。因此，除了一定数量的HNF1alpha、HNF1beta、HNF3alpha、HNF3beta和HNF4转录本的共表达以及C/EBPdelta的抑制外，HNF3gamma、C/EBPbeta和C/EBPbeta的表达可能是肝细胞获得高度分化功能所必需的。

A small hepatocyte can clonally proliferate for more than 3 months when the cells are cultured in DMEM supplemented with 10% FBS,10mM nicotinamide, 1mM ascorbic acid 2-phosphate, 10ng/mI EGF and 1% DMSO.The cell divides to form a colony and the number of the cells reaches to more than 100 cells within 20 days. With time in culture, cells with a large cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with a crystaline nucleoid, which are typical mature heatocytes. Immunoreactivity to Cx32 and well-developed bile canaliculus structures are often observed between cells. Thus, we suggest that small hepatocytes may be considered as "committed progenitor cells" that can further differentiate into mature hepatocytes.We established a culture system in which, by adding 2% DMSO to the culture medium after the hepatocytes proliferate, the cells are able to recover differentiated functions such as albumin and transferrin secretion. Although expressions of connexin 32, Cx 26, and tryptophan 2,3-dioxygenase, thought to a highly differentiated function of mature hepatocytes, have never been maintained or re-induced in primary hepatocytes cultured for long period of time by using the conventional culture methods, those mPNA expressions were gradually restored with time after the addition of 2% DMSO and maintained for a month in this culture system.We showed the role of the liver-enriched transcription factors in the transition during which proliferating hepatocytes become quiescent. We found that hepatic differentiation requires not only inhibition of DNA synthesis but also induction of appropriate transcription facors. Thus, expression of HNF3gamma, C/EBPalpha, and C/EBPbeta may be necessary for hepatocytes to acquire highly differentiated functions in addition to coexpression of certain amounts of transcripts of HNF1alpha, HNF1beta, HNF3alpha, HNF3beta, and HNF4 as well as suppression of C/EBPdelta.