K.C. Donnelly Externship - Promotion of Translational/Transdisciplinary Efforts in Graduate and Post-Doctoral Research - Colvin

K.C.

基本信息

批准号：
10381310
负责人：
Robyn L Tanguay
金额：
$ 1.49万
依托单位：
OREGON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-23 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10381310
关键词：
3-Dimensional Aromatic Polycyclic Hydrocarbons Biological Assay Cell Culture Techniques Cell Line Cells Competence Cultured Cells DNA Adducts DNA Damage DNA Sequence Alteration Detection Diesel Exhaust Electrophoresis Enzymes HepG2 Human Image In Vitro Inhalation Exposure Learning Lung Malignant Neoplasms Mediating Metabolic Methods Modeling Nucleotide Excision Repair Phase Postdoctoral Fellow Process Protocols documentation Reporting Research Techniques Toxic effect Training Wood material airway epithelium bronchial epithelium cigarette smoking computerized data processing exposure route externship metabolic abnormality assessment tumor growth

3-Dimensional Aromatic Polycyclic Hydrocarbons Biological Assay Cell Culture Techniques Cell Line Cells Competence Cultured Cells DNA Adducts DNA Damage DNA Sequence Alteration Detection Diesel Exhaust Electrophoresis Enzymes HepG2 Human Image In Vitro Inhalation Exposure Learning Lung Malignant Neoplasms Mediating Metabolic Methods Modeling Nucleotide Excision Repair Phase Postdoctoral Fellow Process Protocols documentation Reporting Research Techniques Toxic effect Training Wood material airway epithelium bronchial epithelium cigarette smoking computerized data processing exposure route externship metabolic abnormality assessment tumor growth

展开

项目摘要

PROJECT DESCRIPTION In brief, polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion processes such as cigarette smoking, diesel exhaust, and wood burning, and some have been associated with several forms of cancer, including lung. PAHs are bioactivated into their reactive metabolites by Phase I metabolizing enzymes in the body to cause DNA damage and mutations leading to tumor growth. The airway epithelium is a primary route of exposure for inhaled PAHs. The use of 3D organotypic culture models are demonstrating promise for use in metabolic studies as compared to traditional in vitro cell culture models that often lack metabolic competency. My proposed project will evaluate the extent of DNA damage mediated by PAH reactive intermediates generated in primary human bronchial epithelial cells cultured in both 2D and 3D organotypic formats as compared to cell lines with reported negative (TK6) and positive (HepG2) results using the newly developed CometChip assay. In particular, we will apply a method for the CometChip that traps single strand breaks formed during nucleotide excision repair enabling detection of PAH-bulky DNA adducts. This project will aid in describing the relevance of the 2D and 3D organotypic models for toxicity studies relating to DNA damage induced by PAH metabolites. The specific objectives of this project are to 1) obtain training in CometChip assay techniques such as chip formation, cell loading, electrophoresis, and imaging protocols using TK6 and HepG2 cells 2) obtain training needed for processing data collected from the CometChip assay and 3) learn to adapt protocols as needed to utilize the assay for 2D and 3D primary NHBE culture models with PAH exposures.

项目描述简而吸烟，柴油排气和燃烧木材，有些与几种形式有关癌症，包括肺。通过I期代谢酶将PAH的生物活化入其反应性代谢物中在体内导致DNA损伤和突变导致肿瘤生长。气道上皮是主要的吸入PAH的暴露途径。 3D器官培养模型的使用证明了对与传统的体外细胞培养模型相比，用于代谢研究，这些模型通常缺乏代谢能力。我提出的项目将评估PAH反应性介导的DNA损伤的程度在2D和3D器官中培养的原代人支气管上皮细胞中产生的中间体格式与使用新近的细胞系（TK6）和阳性（HEPG2）相比开发的cometchip分析。特别是，我们将应用一种方法来捕获单链的cometchip 在核苷酸切除修复过程中形成的断裂，可检测PAH卵形DNA加合物。这个项目将有助于描述与DNA有关的毒性研究2D和3D器官模型的相关性 PAH代谢产物引起的损害。该项目的具体目标是1）接受培训 cometchip测定技术，例如芯片形成，电池负荷，电泳和成像方案使用TK6和HEPG2细胞2）获得从Cometchip分析收集的数据所需的培训 3）学会根据需要调整协议，以利用2D和3D主要NHBE培养模型的测定法 PAH暴露。