Leukocyte-Endothelial Cell Interaction in Aging

描述(由申请人提供)：趋化因子生物学已经成为许多重要的免疫和炎症过程的中心。然而，人们对衰老对T细胞趋化因子功能的影响知之甚少。这项建议的具体目标是检验这一假设，即衰老与T细胞C-C趋化因子受体表达增加有关，这是由C-C趋化因子受体启动子低甲基化引起的。 具体目标1将检验假设的第一部分，即衰老与T细胞C-C趋化因子受体表达增加有关。衰老对T细胞C-C和C-X-C趋化因子受体表达和功能的影响将定义为1.RNA水平(通过微阵列基因芯片技术筛选并经核糖核酸酶保护分析(RPA)证实)。2.蛋白质水平(蛋白质印迹)。3.功能水平(体外黏附和趋化实验及体内小鼠趋化因子依赖的皮肤炎症模型)。此外，还将测定细胞因子刺激后的T细胞趋化因子反应。 具体目标2将检验假设的后半部分，即启动子低甲基化是导致衰老相关的T细胞C-C趋化因子受体基因表达增加的机制。CCR5基因在转录水平的调控将通过RNA降解研究和体外转录核连续体实验来证实。转录因子在CCR5基因表达调控中的作用将通过使用启动子结构和荧光素酶/β-半乳糖苷酶报告系统的转基因研究来排除。将进行亚硫酸氢盐测序，以确定整个生命周期内T细胞的启动子甲基化状态。将对CCR5启动子进行“补丁”甲基化，以确定DNA甲基化的效果。最后，DNA甲基转移酶和相关调控蛋白的表达将通过RPAs和Western blotts来确定。

DESCRIPTION (provided by applicant): Chemokine biology has emerged as central to many important immune and inflammatory processes. However, very little is known about the effect of aging on T cell chemokine function. The specific goal of this proposal is to test the hypothesis that aging is associated with increased T cell C-C chemokine receptor expression that is caused by C-C chemokine receptor promoter hypomethylation. Specific Aim 1 will test the first part of the hypothesis that aging is associated with increased T cell C-C chemokine receptor expression. The effects of aging on T cell C-C and C-X-C chemokine receptor expression and function will be defined at 1. The RNA level (screening by microarray GeneChip technology and confirmed by ribonuclease protection assays (RPAs)). 2. The protein level (Western blot). 3. The functional level (in vitro adhesion and chemotactic assays and in vivo murine chemokine-dependent cutaneous inflammation model). In addition, T cell chemokine response following cytokine stimulation will be determined. Specific Aim 2 will test the second half of the hypothesis that promoter hypomethylation is the mechanism responsible for aging-associated increased in T cell C-C chemokine receptor gene expression. Regulation of CCR5 gene at the transcriptional level will be confirmed by RNA degradation studies and In vitro transcription nuclear run-on assay. The role of transcription factors in the regulation of CCR5 gene expression will be excluded by transfection studies using promoter constructs and the luciferase/beta- galactosidase reporter system. Bisulfite sequencing will be done to determine the promoter methylation status of T cells from across the life span. "Patch" methylation of the CCR5 promoter will be done to determine the effect of DNA methylation. Finally, expression of DNA methyltransferases and the related regulatory proteins will be determined by RPAs and Western blots.