Engineered Platforms to Manipulate Intracellular Redox

操纵细胞内氧化还原的工程平台

• 批准号: 7230234
• 负责人: Paul J. A. Kenis
• 金额: $ 17.82万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7230234
• 关键词: Address; Affect; Apoptosis; Biocompatible; Biological; Biosensor; Cell Cycle Progression; Cell Line; Cell Proliferation; Cell physiology; Cells; Chemistry; Complex; Condition; Contact Inhibition; Cultured Cells; DNA biosynthesis; DNA chemical synthesis; Disruption; Electrodes; Engineering; Environment; Exhibits; Fibroblasts; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Genetic; High Pressure Liquid Chromatography; Homeostasis; Human; Imagery; Individual; Lead; Life; Measures; Mediating; Methods; Molecular; Molecular Biology; Monitor; Mutation; One-Step dentin bonding system; Organelles; Oxidation-Reduction; Pathway interactions; Public Health; Regulation; Research; Research Personnel; Role; Signal Pathway; Signal Transduction; Time; Transduction Gene; Visual; base; cell growth; density; design; fibrosarcoma; novel; oxidation; physical science; programs; response; sensor; systems research; tumorigenesis; uncontrolled cell growth

DESCRIPTION (provided by applicant): In redox systems research domains that traditionally belong to the physical sciences, chemistry and molecular biology are coming together to offer new synergistic opportunities for understanding and manipulating basic cellular processes that underlie complex biomedical problems (e.g., tumorigenesis). Parallel with this recognition emerges that intracellular redox status exerts influence on the normal cellular processes of DNA synthesis, selective gene expression, cell cycle progression, proliferation, differentiation, and apoptosis. However, molecular mechanisms mediating redox sensitivity are still poorly defined. Current pharmacological methods to alter intracellular redox potential require significant manipulation of culture conditions that perturb intracellular homeostasis. To overcome this problem and to answer fundamental questions concerning intracellular redox and cell growth, this proposal focuses on the creation of engineered electrochemical platforms that will enable precise manipulation of intracellular redox and novel genetic constructs that will enable real-time and extended assessment of alterations in intracellular redox without cellular disruption. Equipped with cell study platforms and biosensors for visualization (SA 1 & 2), we will address a central cell biological question of primary biomedical relevance that being the relationship between intracellular redox and density-dependent contact inhibition of cell growth (SA 3). The proposed research will thus aid public health by aiming to unravel the role of intracellular redox in uncontrolled cell growth (i.e. tumorigenesis). Specific Aim 1: Design and validate engineered electrochemical (EEC) platforms for cell studies that permit precise control of the intracellular redox environment. We will measure intracellular and intraorganellar redox state as a function of externally applied potential by monitoring the ratios of redox-active species (GSH/GSSG) and with fluorescence microscopy using markers for GSH and ROS, as well as novel gene constructs. Specific Aim 2: Develop and validate FRET biosensors that permit visual monitoring of intracellular and intraorganellar redox potentials. The envisioned genetic constructs encoding FRET-based redox sensors will be stably transfected into target cells allowing real-time monitoring of intracellular redox potentials in live cells. Incorporating organelle-specific targeting sequences will permit the monitoring of intraorganellar redox potential. Specific Aim 3: Use EEC platforms and FRET biosensors to determine how reversibly manipulating intracellular redox status affects cell growth in non-transformed and transformed human fibroblast cell lines. Nontransformed cells become increasingly oxidized concurrent with density-dependent contact inhibition. Thus, we hypothesize that mutations in redox-regulated signaling pathways that render cells unable to initiate contact inhibition may contribute to tumorigenesis. This hypothesis will be addressed with the EEC platforms (and FRET Biosensors for visualization) by reversibly manipulating the intracellular redox status of nontransformed IMR-90 human fibroblasts versus HT-1080 human fibrosarcoma cells (do not exhibit contact inhibition) to determine whether and how progressive oxidation of the intracellular environment contributes to density-dependent contact inhibition.

描述（由申请人提供）：在传统上属于物理科学的氧化还原系统研究领域中，化学和分子生物学聚集在一起，为理解和操纵基本的基本细胞过程提供了新的协同机会，这些过程基于复杂的生物医学问题（例如，肿瘤生物发生）。与这种识别的平行表明，细胞内氧化还原状态对DNA合成，选择性基因表达，细胞周期进程，增殖，分化和凋亡的正常细胞过程作用。但是，介导氧化还原敏感性的分子机制仍然很差。当前改变细胞内氧化还原电位的药理方法需要对扰动细胞内稳态的培养条件进行重大操纵。为了克服这个问题并回答有关细胞内氧化还原和细胞生长的基本问题，该提案着重于创建工程的电化学平台，这些平台将能够精确地操纵细胞内氧化还原和新型遗传构建体，这些遗传构建体和新型遗传构建体将实现实时和扩展细胞内氧化还原的变化的评估，而无需蜂窝外部氧化还原。配备了可视化的细胞研究平台和生物传感器（SA 1＆2），我们将解决主要生物医学相关性的中央细胞生物学问题，即细胞内氧化还原与密度依赖性依赖性接触细胞生长之间的关系（SA 3）。因此，拟议的研究将通过旨在揭示细胞内氧化还原在不受控制的细胞生长中的作用（即肿瘤发生）来帮助公共卫生。特定目的1：设计和验证工程的电化学（EEC）平台，用于允许精确控制细胞内氧化还原环境的细胞研究。我们将通过监测氧化还原活性物种（GSH/GSSG）的比率以及使用标记GSH和ROS的标记以及新的基因构建体来测量细胞内和辅助氧化还原状态，这是外部应用潜力的函数。特定目标2：开发和验证允许对细胞内和卫生层氧化还原电位的观察监测的FRET生物传感器。编码基于FRET的氧化还原传感器的设想遗传构建体将稳定转染到靶细胞中，从而实时监测活细胞中细胞内氧化还原电位。结合特定于细胞器的靶向序列将允许监测田内氧化还原电位。特定目标3：使用EEC平台和FRET生物传感器来确定如何可逆地操纵细胞内氧化还原状态会影响未转化和转化的人成纤维细胞细胞系的细胞生长。非转化的细胞与密度依赖性接触抑制越来越多地氧化。因此，我们假设氧化还原调节的信号通路中的突变使细胞无法启动接触抑制可能有助于肿瘤发生。通过可逆地操纵非转换IMR-90人类成纤维细胞的细胞内氧化还原状态与HT-1080人类纤维肉瘤细胞（不表现出抑制与内部依赖的环境依赖的环境），该假设将与EEC平台（以及可视化的FRET生物传感器）一起解决。