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

在含10%FBS、10 mM烟酰胺、1 mM抗坏血酸2-磷酸盐、10 ng/ml EGF和1%DMSO的DMEM培养基中培养3个月以上，细胞分裂形成集落，20天内细胞数达100个以上。随着培养时间的推移，具有大细胞质的细胞出现在集落中。它们有许多线粒体和大的过氧化物酶体，具有结晶状的类核，是典型的成熟热细胞。在细胞之间经常观察到对Cx 32的免疫反应性和发育良好的胆小管结构。因此，我们认为，小肝细胞可以被认为是“定向祖细胞”，可以进一步分化为成熟的肝细胞。我们建立了一个培养系统，在该系统中，通过添加2%DMSO的肝细胞增殖后的培养基中，细胞能够恢复分化的功能，如白蛋白和转铁蛋白分泌。虽然连接蛋白32、Cx 26和色氨酸2，3-双加氧酶的表达被认为是成熟肝细胞的高度分化功能，但在使用常规培养方法长时间培养的原代肝细胞中从未被维持或再诱导，在加入2%~ 30%的磷酸盐后，随着时间的推移，mPNA的表达逐渐恢复结果表明，肝细胞中富含的转录因子在增殖肝细胞向静止肝细胞转变过程中起重要作用。我们发现肝细胞分化不仅需要抑制DNA合成，而且需要诱导适当的转录因子。因此，除了共表达一定量的HNF 1 α、HNF 1 β、HNF 3 α、HNF 3 β和HNF 4的转录物以及抑制C/EBP δ外，HNF 3 γ、C/EBP α和C/EBP β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.