Leukocyte-Endothelial Cell Interaction in Aging

描述（由申请人提供）：趋化因子生物学已成为许多重要免疫和炎症过程的核心。然而，关于衰老对T细胞趋化因子功能的影响知之甚少。该提案的具体目标是检验衰老与T细胞C-C趋化因子受体表达增加相关的假设，该表达增加是由C-C趋化因子受体启动子低甲基化引起的。 具体目标1将测试假设的第一部分，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在1. RNA水平（通过微阵列基因芯片技术筛选并通过核糖核酸酶保护测定（RPA）确认）。2.蛋白质水平（Western blot）。3.功能水平（体外粘附和趋化性试验和体内鼠趋化因子依赖性皮肤炎症模型）。此外，将确定细胞因子刺激后的T细胞趋化因子应答。 特异性目标2将检验启动子低甲基化是负责衰老相关的T细胞C-C趋化因子受体基因表达增加的机制这一假设的后半部分。将通过RNA降解研究和体外转录核运行试验证实CCR 5基因在转录水平的调控。通过使用启动子构建体和荧光素酶/β-半乳糖苷酶报告系统的转染研究，将排除转录因子在CCR 5基因表达调控中的作用。将进行亚硫酸氢盐测序以确定整个寿命期内T细胞的启动子甲基化状态。将进行CCR 5启动子的“补丁”甲基化以确定DNA甲基化的影响。最后，DNA甲基转移酶和相关调节蛋白的表达将通过RPA和蛋白质印迹来确定。

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.