Tirnitz-Parker Janina E E, Tonkin Joanne N, Knight Belinda, Olynyk John K, Yeoh George C T
School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley, Australia.
Int J Biochem Cell Biol. 2007;39(12):2226-39. doi: 10.1016/j.biocel.2007.06.008. Epub 2007 Jun 29.
Oval cells have great potential for use in cell therapy to treat liver disease, however this cannot be achieved until the factors which govern their proliferation and differentiation are better understood. We describe a method to establish primary cultures of murine oval cells, and the derivation of two novel lines from these. Primary cultures from the livers of wildtype or TAT-GRE lacZ transgenic mice subjected to a choline-deficient, ethionine-supplemented diet comprised up to 80% oval cells at day 7 based on A6 or CK19 staining. Cell lines were clonally derived, which underwent spontaneous immortalisation following prolonged maintenance in culture. Immunostaining and RT-PCR demonstrated they express hepatocytic and biliary markers and they were therefore termed "bipotential murine oval liver" (BMOL) cells. Under proliferating culture conditions, BMOL or BMOL-TAT cells abundantly expressed oval cell and biliary markers, whereas mature hepatocytic markers were upregulated when the growth conditions were changed to facilitate differentiation. Hepatic differentiation of BMOL-TAT cells could be traced by measuring the expression of their lacZ transgene, which is driven by a promoter element from tyrosine aminotransferase (TAT), a marker of adult hepatocytes. Interestingly, haematopoietic markers were upregulated in superconfluent cultures, indicating a possible multipotentiality. None of the cell lines grew in semi-solid agar, nor did they form tumours in nude mice, suggesting they are non-tumourigenic. These novel murine oval cell lines, together with a reliable method for isolation and culture of primary oval cells, will provide a useful tool for investigating the contribution of oval cells to liver regeneration.
卵圆细胞在用于治疗肝脏疾病的细胞疗法中具有巨大潜力，然而，在更好地了解调控其增殖和分化的因素之前，这一目标无法实现。我们描述了一种建立小鼠卵圆细胞原代培养物的方法，以及从这些培养物中衍生出两个新细胞系的过程。对野生型或TAT-GRE lacZ转基因小鼠肝脏进行胆碱缺乏、添加乙硫氨酸的饮食处理后，在第7天基于A6或CK19染色，原代培养物中卵圆细胞含量高达80%。细胞系是通过克隆获得的，在长期培养后会自发永生化。免疫染色和逆转录-聚合酶链反应表明它们表达肝细胞和胆管标志物，因此被称为“双潜能小鼠卵圆肝细胞”（BMOL）细胞。在增殖培养条件下，BMOL或BMOL-TAT细胞大量表达卵圆细胞和胆管标志物，而当生长条件改变以促进分化时，成熟肝细胞标志物会上调。BMOL-TAT细胞的肝分化可以通过测量其lacZ转基因的表达来追踪，该转基因由成年肝细胞标志物酪氨酸转氨酶（TAT）的启动子元件驱动。有趣的是，在汇合过度的培养物中造血标志物会上调，表明可能具有多潜能性。这些细胞系在半固体琼脂中均不生长，在裸鼠中也不形成肿瘤，表明它们不具有致瘤性。这些新型小鼠卵圆细胞系，连同分离和培养原代卵圆细胞的可靠方法，将为研究卵圆细胞对肝脏再生的作用提供有用的工具。
