ORGANOTYPIC LIVER CULTURES AND HEPATOCARCINOGENESIS

描述(由申请人提供)： 肝癌的发生是一个复杂的过程，需要在构成哺乳动物肝脏的三维组织结构内进行细胞间的相互作用。我们寻求通过将这一过程尽可能地扩展到培养皿来提高我们研究肝癌发生的能力。我们已经建立了一种器官型大鼠肝脏模型，在该模型中，活组织在培养中保持了超过90天。这项应用的总体目标是进一步发展这一大鼠肝脏器官型培养模型(未来也包括人类肝肿瘤组织)，并证明其在研究胆碱缺乏的大鼠肝癌发生中的有效性。为此，我们提出了三个具体目标：(1)对于我们的器官型大鼠肝培养模型，我们将分析和测试所有培养基组分，直到我们得出一个定义的培养基组分，通过双重生命染色、DNA阵列揭示的基因表达模式、通过酶的数量和活性揭示的代谢能力来判断该培养基能提供长时间的活性。(2)我们将进一步鉴定CWSV-1细胞的特性，CWSV-1细胞在培养中形成与正常肝脏相似的组织岛，特别是在其产生多种肝细胞类型的能力以及在胆碱缺乏的条件下向肿瘤细胞的发展方面。(3)我们将在AIMS(1)和(2)中比较这两种培养模型的PBN和4-OH-PBN促进细胞凋亡和抑制肿瘤生长的能力。最终，我们寻求一种器官类型的培养模型，在该模型中，可以操纵从肿瘤起始到进展到完全转移的癌症发生的全过程，并对其进行治疗测试。

DESCRIPTION (provided by applicant): Hepatocarcinogenesis is a complex process requiring cell-to-cell interactions within the three-dimensional tissue architecture that constitutes the mammalian liver. We seek to enhance our abilities to investigate hepatocarcinogenesis by extending the process to the culture dish as much as possible. We have developed an organotypic rat liver model in which viable tissue has been maintained in culture for longer than 90 days. The overall objective for this application is to further develop this organotypic culture model for rat liver (and in the future, for human liver tumor tissue, as well), and to demonstrate its usefulness for investigating choline-deficient hepatocarcinogenesis in a rat model. Toward this end, we propose three Specific Aims: (1) For our organotypic rat liver culture model, we will analyze and test all medium components until we arrive at a defined medium that provides long periods of viability as judged by dual vital staining, by gene expression patterns revealed by DNA arrays, by metabolic capabilities revealed by enzyme amounts and activities. (2) We will further characterize CWSV-1 cells, which form islands of tissue in culture that approximate normal liver in pattern of organization, especially as to their ability to give rise to more than one liver cell type and their progression to tumorigenic cells in choline-deficient medium. (3) We will compare and contrast these two culture models in Aims (1) and (2) with respect to the ability of PBN and 4-OH-PBN to increase apoptosis and inhibit tumor development. Ultimately, we seek an organotypic culture model in which the full process of carcinogenesis from tumor initiation through progression to full metastatic cancer can be manipulated and cures tested.