Innovative Toxicology Models for Drug Evaluation

药物评价的创新毒理学模型

• 批准号: 6652368
• 负责人: Jiing-Kuan YEE
• 金额: $ 17.5万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6652368
• 关键词: biochemistry; cell differentiation; cell line; cell proliferation; cisplatin; complementary DNA; cyclophosphamide; drug adverse effect; drug screening /evaluation; hepatotoxin; integrase; kidney function; liver cells; methotrexate; paclitaxel; renal toxin; renal tubule; technology /technique development; telomerase; transport proteins

DESCRIPTION (provided by applicant): A. Specific Aims: The overall goal of this proposal is to use reversible immortalization approach to establish cell lines derived from primary human renal proximal tubule epithelial cells (RPTECs) and hepatocytes. Liver and kidney are two critical organs for drug metabolism and excretion, and are frequently affected by drug-induced toxicity. Currently, no suitable human kidney cell line is available for drug screening in the early stages of drug discovery and development. While primary human hepatocytes are available, the high cost to culture such cells precludes their use in the drug screening process. If immortalized cell lines that are phenotypically stable and retain all or most of the primary cell functions can be established, they can be used for high-throughput screening of potential drugs. There are three specific aims in this proposal. I. Characterization of TH1 and TH7 cells for kidney functions: In our preliminary study, we used human immunodeficiency virus (HIV)-based vectors containing the cDNAs encoding the SV40 T antigen (Tag) and the catalytic subunit of human telomerase (hTERT) to establish several immortalized cell lines derived from primary human RPTECs. We propose to characterize the biochemical and functional activities in two of these cell lines, TH1 and TH7. We will determine the basal expression and the activity of xenobiotic metabolizing enzymes in these two cell lines. We will evaluate the activity of drug transport proteins, drug uptake and efflux. We also propose to use Cre-LoxP mediated recombination system to remove the introduced Tag and the hTERT cDNAs from these two cell lines and examine its effect on cell proliferation, differentiation and biochemical functions. II. Establishment and characterization of immortalized human hepatocytes: We propose to use the same strategy as described in specific aim I to establish and characterize immortalized human hepatocytes. Ill. Evaluation of drug toxicity in the immortalized cell lines: We will determine the drug sensitivity of the established cell lines, including methotrexate (MTX), cisplatin, cyclophosphamide and paclitaxel. The toxicity induced by these drugs in primary human cells and in cell lines derived from hepatocytes and RPTECs will also be examined and the results will be compared with those from the immortalized cell lines established in this current study.

描述（由申请人提供）： A. 具体目标： 该提案的总体目标是使用可逆永生化方法建立源自原代人肾近端小管上皮细胞（RPTEC）和肝细胞的细胞系。肝脏和肾脏是药物代谢和排泄的两个关键器官，经常受到药物毒性的影响。目前，尚无合适的人肾细胞系可用于药物发现和开发早期阶段的药物筛选。虽然可以使用原代人肝细胞，但培养此类细胞的高成本阻碍了它们在药物筛选过程中的使用。如果能够建立表型稳定并保留全部或大部分原代细胞功能的永生化细胞系，它们可用于潜在药物的高通量筛选。该提案有三个具体目标。 I. TH1 和 TH7 细胞肾功能的表征： 在我们的初步研究中，我们使用基于人类免疫缺陷病毒（HIV）的载体，包含编码SV40 T抗原（Tag）和人类端粒酶（hTERT）催化亚基的cDNA，建立了几种源自原代人类RPTEC的永生化细胞系。我们建议表征其中两种细胞系 TH1 和 TH7 的生化和功能活性。我们将确定这两种细胞系中外源代谢酶的基础表达和活性。我们将评估药物转运蛋白的活性、药物摄取和流出。我们还建议使用Cre-LoxP介导的重组系统从这两种细胞系中去除引入的Tag和hTERT cDNA，并检查其对细胞增殖、分化和生化功能的影响。 二.永生化人肝细胞的建立和表征： 我们建议使用与特定目标 I 中描述的相同策略来建立和表征永生化人类肝细胞。 III.永生化细胞系中药物毒性的评价： 我们将确定已建立的细胞系的药物敏感性，包括甲氨蝶呤（MTX）、顺铂、环磷酰胺和紫杉醇。还将检查这些药物在原代人类细胞以及源自肝细胞和 RPTEC 的细胞系中诱导的毒性，并将结果与​​本研究中建立的永生化细胞系的结果进行比较。