REGULATION OF AFP GENE EXPRESSION IN HEPATOMA CELLS IN VITRO AND IN VIVO

这项研究的长期目标是了解 在发育和肿瘤形成过程中的差异基因表达;特别是 AFP基因在肝细胞中的表达/抑制机制。 我们 我将继续研究这一假说，即这种调节是由 体液因子与转录因子的相互作用。 也我们 我会问，正常的发展建立的控制机制， 在肿瘤转化过程中发生改变。 我们建议定义参与AFP诱导的机制 在正常成人肝细胞和最低限度异常肝细胞中的表达 肝癌细胞株OR-HEPA。 成年C，H小鼠将 用250万个细胞接种IP以产生这种腹水形式， 肿瘤 两周后，宿主小鼠将注射L-乙醇胺， （0.5 μ g/体重）发现在成人肝脏中诱导AFP。 该实验 在我们早期的研究中开发的模型允许比较分析 在相同实验条件下对正常和转化细胞的影响 条件 甲胎蛋白在宿主肝及腹水中的表达 细胞，24-96小时后，将与其他肝脏的表达相关 通过放射免疫化学和斑点/北方印迹检测Trf和Alb基因。 将使用核连续测定来评估以下转录水平： 一组基因，包括癌基因（c-fos、c-Ha-ras、c-jun）和生长 因子（IGF-II、TGF-β、TGF-α）。 此外，来自 两种组织都将通过与AFP小基因结合，然后通过凝胶电泳进行检查。 转移迁移率和DNaseI保护试验检测特异性反式作用 因素 为了比较胎儿核蛋白的类似实验 会完成的 使用动物模型更好地了解基本的细胞机制 与人类健康问题相关的是一种公认的实验方法。 通过这次调查获得的知识将有助于澄清 癌细胞和其他细胞/遗传的病因学和特征 人类疾病，因此具有潜在的治疗意义， 目的 该项目的另一个主要目标是继续为学生提供一个 最新的培训和指导，在重要领域的蜂窝和 生物医学科学的分子生物学

The long term goal of this research is to understand the regulation of differential gene expression during development and neoplasia; specifically the expression/repression mechanism of the AFP gene in liver cells. We will continue working on the hypothesis that this regulation is mediated by the interaction of humoral factors with transcription factors. Also we will ask whether the normal developmentally established control mechanism is altered during neoplastic transformation. We propose to define the mechanism involved in the induction of AFP expression in normal adult liver cells and in the minimally deviant hepatoma cells, OR-HEPA established in this lab. Adult C,H mice will be inoculated IP with 2.5 million cells to produce the ascites form of this tumor. After two weeks, host mice will be injected with L-ethionine (0.5mug/body weight) found to induce AFP in adult liver. This experimental model developed in our earlier studies allows comparative analysis of the effects on normal and transformed cells under the same experimental condition. The expression of AFP in both the host liver and the ascites cells, after 24-96 hr, will be correlated to expression of other liver genes (Trf and Alb) by radio-immunochemistry and dot/northern blots. Nuclear run-on assays will be used to assess the transcriptional levels of a battery of genes including oncogenes (c-fos, c-Ha-ras, c-jun) and growth factors (IGF-II, TGF-beta, TGF-alpha). Additionally, nuclear proteins from both tissues will be examined by binding to AFP mini-genes followed by gel shift mobility and DNaseI protection assays to detect specific trans-acting factors. For comparison similar experiments with fetal nuclear proteins will be done. The use of animal models to better understand basic cellular mechanisms related to human health problems is a well accepted experimental approach. The knowledge obtained through this investigation will help to clarify the etiology and characteristics of the cancer cell and other cellular/genetic human diseases and thus, has potential implications for therapeutic purposes. Another main goal of this project is to continue offering the students an up-to-date training and guidance in the important areas of cellular and molecular biology of biomedical sciences.