Cell proliferation in models of fibrosis

纤维化模型中的细胞增殖

基本信息

批准号：
6901794
负责人：
NICHOLAS H HEINTZ
金额：
$ 23.76万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-06-04 至 2006-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6901794
关键词：
artificial chromosomes cell cycle cell proliferation cyclins disease /disorder model enzyme activity free radical oxygen gene induction /repression gene targeting genetically modified animals green fluorescent proteins laboratory mouse mitogen activated protein kinase pollution related respiratory disorder pulmonary fibrosis /granuloma respiratory epithelium synchronous cell division tissue /cell culture

项目摘要

Progression through G1 and entry into the S phase are carefully regulated during the eukaryotic cell cycle. Induction of cell proliferation involves several sequential steps that include: 1) activation of mitogen-activated protein kinases (MAPK), 2) expression of early response genes such as c- fos and c-jun, 3) transcriptional licensing and S phase entry. Asbestos, reactive oxygen, and reactive nitrogen species (ROS/RNS) influence progression through G1 and entry into the S phase by activating or perturbing MAPK cascades, altering the activity of G1 cyclins or alt4ering the activation of E2F. Here, we propose specific aims to examine progression through G1 and S phase entry in models of allergic airway hyperresponsiveness/fibrosis and asbestosis. First, we will document the relationship between cell cycle progression and patterns of MAPK activation, expression of cyclin D1, and origin licensing by Cdc6 in synchronized murine alveolar type II (C1) cells in response to asbestos, RO2 and cationic proteins. Second, we will use homologous recombination of bacterial artificial chromosomes (BACs) to generate alleles of cyclin D1 and cfdc6 that contain internal ribosome entry sites (IRES)-enhanced green fluorescent protein (EGFP) expression cassettes in the 3' untranslated regions of the genes. Using a novel gene transfer technique, these alleles will be transferred into cells in cultured, and regulated expression during the cell cycle of dicistonic mRNAs from the BAC alleles will be assessed. Third, those BAC alleles displaying proper expression of dicistonic mRNAs encoding cyclin D1-IRES-EGFP and Cdc6-IRES-EGFP will be used to generate transgenic reporter mice. Mice bearing these transgenic BAC alleles will be used to study progression through G1 and commitment to S phase in epithelial cells in inhalation models used in projects 1-3. Finally, backcrossing of BAC transgenic mice with mice expressing dominant negative MEK1 will be used to define the role of ERKs in governing progression through G1 and S phase entry, as well as their relationship to the development of epithelial cell proliferation and fibrosis.

在真核细胞周期中，通过G1的进展和进入S相的进入。细胞增殖的诱导涉及几个顺序步骤，其中包括：1）激活有丝分裂原激活的蛋白激酶（MAPK），2）早期反应基因（例如C-FOS和C-JUN，3）的表达，3）转录许可和S相进入。石棉，活性氧和反应性氮种（ROS/RNS）通过激活或扰动MAPK级联反应，通过G1进入S相的进展，改变G1细胞周期蛋白的活性或Alt4激活E2F的活性。在这里，我们提出的特定目的是在过敏性气道高反应/纤维化和石棉病模型中通过G1和S相进入的进展。首先，我们将记录细胞周期进程与MAPK激活的模式，细胞周期蛋白D1的表达以及Cdc6在同步的鼠肺泡II型（C1）细胞中响应石棉，RO2和阳离子蛋白的同步鼠肺泡II（C1）细胞之间的关系。其次，我们将使用细菌性人造染色体（BAC）的同源重组来产生含有内部核糖体进入位点（IRES）增强的绿色荧光蛋白（EGFP）表达盒的细胞周期蛋白D1和CFDC6等位基因。使用一种新颖的基因转移技术，将在培养的细胞中转移到细胞中，并将评估来自BAC等位基因的双质mRNA细胞周期中的调节表达。第三，将使用编码Cyclin D1-IRES-EGFP和CDC6-IRES-EGFP的DICistonic mRNA正确表达的BAC等位基因来产生转基因报告者小鼠。具有这些转基因BAC等位基因的小鼠将用于通过G1研究进展，并在项目1-3中使用的吸入模型中上皮细胞中的S相承诺。最后，用表达显性负MEK1的小鼠对BAC转基因小鼠的反向交叉将定义ERK在通过G1和S相进入的过程中的作用，以及它们与上皮细胞增殖和纤维化发展的关系。