ANALYSIS OF IN VITRO DIFFERENTIATION OF A HUMAN HEPATOMA

人肝癌细胞系Hep G2在体外分化，从 胎肝的表型特征与更接近的表型 成年人。这种区别表现为戏剧性地延长了 培养时间翻倍，合成能力下降 甲胎蛋白、醛缩酶A、C和丙酮酸激酶K的增加 白蛋白、醛缩酶B和丙酮酸激酶L的合成 描述了在此过程中对白蛋白基因的详细分析 差异化。信使RNA水平和转录变化将是 用Northern blotts、核径流实验和定量 溶液杂交法。这项研究的第二阶段涉及 杂合基因的构建及其在Hep G2细胞中的表达 负责分化相关改变的序列。瞬变 将使用假定的肝脏特异性进行表达分析 附在CAT上的调节序列，一种细菌基因，将被用作 记号笔。这应该允许相当快速地描述序列 白蛋白表达所必需的。互补性实验将监测 具有可变拷贝的稳定转染体中杂交基因的转录 数。这项工作的第三阶段将使用细胞自由转录 从亚克隆Hep G2中提取的蛋白质鉴定 肝脏特异性白蛋白基因的转录因子 表情。

The human hepatoma line Hep G2 differentiates in vitro, progressing from a phenotype characteristic of fetal liver to one which more closely resembles the adult. This differentiation is denoted by a dramatic lenthening of the doubling time of the cultures, a decline in the synthesis of alphafetoprotein, aldolase A, C and pyruvate kinase K and an increase in the synthesis of albumin, aldolase B and pyruvate kinase L. This proposal describes a detailed analysis of the albumin gene during this differentiation. Messenger RNA levels and changes in transcription will be measured using Northern blots, nuclear run off experiments and quantitative solution hybridization assays. A second phase of this study involves the construction of hybrid genes and transfection into Hep G2 to define the sequences responsible for differentiation dependent alterations. Transient expression assays will be carried out using putative liver specific regulatory sequences attached to CAT, a bacterial gene to be used as a marker. This should allow a reasonably rapid delineation of the sequences necessary for albumin expression. Complementary experiments will monitor transcription at the hybrid gene in stable transfectants with varying copy number. A third phase of this work will use cell free transcription extracts prepared from Hep G2, a subclone, to identify protein transcriptional factors responsible for liver specific albumin gene expression.