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

肝组织体外重建研究：人工肝脏的应用。

基本信息

批准号：
08670260
负责人：
MITAKA Toshihiro
金额：
$ 1.47万
依托单位：
Sapporo Medical University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

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.

当在补充10％FBS，10mm烟酰胺，1mm抗坏血酸2-磷酸盐，10NG/MI EGF和1％DMSO的DMEM中培养细胞，在DMEM中培养细胞时，小肝细胞可以在克隆上增殖3个月以上。并且细胞的数量在20天内达到了100多个细胞。随着培养时间的时间，具有较大细胞质的细胞出现在一个菌落中。它们具有许多线粒体和大型过氧化物酶体，具有晶体核苷，它们是典型的成熟热组织。在细胞之间经常观察到对CX32和发达的胆管结构的免疫反应性。因此，我们建议小型肝细胞可以被视为“承诺的祖细胞”，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，在这种培养系统中，通过在肝细胞增殖后向培养基中添加2％DMSO，细胞能够使细胞能够恢复差异化功能，例如白蛋白和转铁蛋白分泌。尽管连接蛋白32，CX 26和色氨酸2,3-二加氧酶的表达是成熟的肝细胞的高度分化功能，但从未在长期培养的原代肝细胞中通过使用常规的常规培养方法来维持或重新诱导或重新诱导过。，在添加2％DMSO后，随着时间的推移逐渐恢复了这些mPNA表达式，并在该培养系统中维持一个月。我们在过渡过程中展示了富含肝脏的转录因子的作用，在此期间，增殖的肝细胞变得静止。我们发现，肝分化不仅需要抑制DNA合成，而且还需要诱导适当的转录设备。因此，HNF3GAMMA，C/EBPALPHA和C/EBPBETA的表达对于肝细胞可能还需要获得高度分化的功能，除了共表达HNF1Alpha的一定量的HNF1Alpha，Hnf1beta，Hnf1beta，hnf3alpha，hnf3alpha，hnf3alpha，hnf3beta，hnf3beta和hnf3beta and Hnf4 /ebpdelta。