Genesis of Liver Carcinomas with Oval Cell Traits

具有卵圆细胞特征的肝癌的起源

• 批准号: 6706247
• 负责人: Douglas Carter Hixson
• 金额: $ 34.27万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6706247
• 关键词: biliary tract neoplasm; carcinogenesis; cell line; cell transplantation; cellular oncology; flow cytometry; gene expression; green fluorescent proteins; hepatocellular carcinoma; laboratory rat; liquid chromatography mass spectrometry; monoclonal antibody; neoplastic transformation; phenotype; preneoplastic state; stem cells; tissue /cell culture

DESCRIPTION:(PROVIDED BY APPLICANT) Oval cells, bipotent cells of ductal origin, are activated in human liver by hepatotoxins, carcinogens, viral agents or chronic disease states, all which greatly increase the risk of liver cancer. A better understanding of oval cells as progenitors of hepatocellular carcinoma (HCC) could lead to valuable insights, particularly if oval cell-derived HCC differ in behavior from their diploid hepatocyte-derived counterparts. The proposed research is structured around the hypothesis that HCC originate, at least in part, from hepatocytes derived de novo from initiated oval cells. These initiated oval cells have newly acquired genetic change(s) that arrest differentiation and produce oval-cell-antigen positive hepatocytes capable of progression to HCC. This hypothesis will be tested by assessing the fate of carcinogen-induced Fischer rat oval cells transplanted via the spleen into the liver of syngeneic hosts with mutated, inactive dipeptidylpeptidase IV (DPPIV). Donor cells will be distinguished histochemically by their expression of active DPPIV or exogenous beta-galactosidase (B-gal). Experiments in Specific Aim I will determine whether established cultures of oval cells induced by ethionine in a choline deficient (CD) diet (CDE) can integrate, differentiate and progress to HCC when promoted by CD diet. To encourage donor cell expansion, host rats will be treated with retrorsine/partial hepatectomy (PH) prior to transplantation. Changes in gene expression following integration will be monitored by phenotyping donor cells at various timepoints after transplantation. In Specific Aim 2, a similar series of experiments will be performed using primary oval cell isolates from DPPIV+ rats maintained on CDE diet or treated with diethylnitrosamine, 2-AAF and PH (resistant hepatocyte regimen). Oval cells will be isolated by high speed FACS using anti Thy-1 antibodies and a unique panel of monoclonal antibodies defining surface lineage markers. HCC will be quantitated by measuring serum DPPIV activity, DPPIV/B-gal activity in liver extracts or by direct quantitation of HCC transections stained for DPPIV, B-gal or known neoplastic markers. In Specific Aim 3, the focus will be on determining if the failure of oval cell and high passage BDEC cultures to upregulate the BDEC antigen, BD.1, following G1/S arrest represents a preneoplastic event. Comparative studies will examine growth characteristics, ductal morphogenesis, adhesion phenotypes, apoptosis and expression of genes activated in the early stages of cholangiocarcinogenesis (c-neu, COX-2, c-met). By elucidating the role of oval cells in liver carcinogenesis, the results from these studies will provide valuable information that can be translated into new strategies for the diagnosis, treatment and prognosis of HCC.

描述：（申请人提供）卵圆细胞，导管双能细胞 在人体肝脏中被肝毒素、致癌物质、病毒因子激活 或慢性疾病状态，所有这些都会大大增加肝癌的风险。 卵圆细胞作为肝癌祖细胞的再认识 (HCC)可能导致有价值的见解，特别是如果卵圆细胞来源的HCC 在行为上与其二倍体肝细胞衍生的对应物不同。的 拟议的研究是围绕肝癌起源的假设， 至少部分来自于从初始卵圆细胞重新衍生的肝细胞。 这些启动的卵圆细胞具有新获得的遗传变化， 分化并产生卵圆细胞抗原阳性肝细胞， 进展为HCC。这一假设将通过评估 致癌物诱导的Fischer大鼠卵圆细胞经脾移植到 具有突变的、失活的二肽基肽酶IV（DPPIV）的同基因宿主的肝脏。 供体细胞将通过它们的活性表达在组织化学上区分。 DPPIV或外源β-半乳糖苷酶（B-gal）。特殊目的实验I 将决定是否建立卵圆细胞培养诱导乙醇 在胆碱缺乏（CD）饮食（CDE）中， 当通过CD饮食促进时进展为HCC。为了促进供体细胞扩增， 宿主大鼠将在给药前用反曲马新/部分肝切除术（PH）处理。 移植整合后基因表达的变化将是 通过对供体细胞进行表型分析来监测， 移植在具体目标2中，将进行一系列类似的实验。 使用CDE上维持的DPPIV+大鼠的原代卵圆细胞分离物进行 饮食或用二乙基亚硝胺、2-AAF和PH（抗性肝细胞）处理 方案）。使用抗Thy-1通过高速FACS分离卵圆细胞 抗体和一组独特的单克隆抗体定义表面谱系 标记。HCC将通过测量血清DPPIV活性、DPPIV/B-gal 肝提取物中的活性或通过直接定量HCC横断 DPPIV、B-gal或已知肿瘤标记物染色。具体目标3中， 重点将是确定卵圆细胞和高传代BDEC的失败是否 在G1/S停滞后上调BDEC抗原BD.1的培养物代表 肿瘤前事件比较研究将检查生长特征， 导管形态发生、粘附表型、细胞凋亡和基因表达 在胆管癌发生的早期阶段被激活（c-neu、考克斯-2、c-met）。 通过阐明卵圆细胞在肝癌发生中的作用， 这些研究将提供有价值的信息， 肝癌的诊断、治疗和预后的策略。