MOLECULAR MECHANISMS OF OXIDANT TOXICITY

衰老是许多致命疾病的最高风险因素。 衰老的自由基理论认为氧化剂毒性在衰老中的作用。 虽然氧化损伤在衰老过程中积累，但尚不清楚氧化剂如何在机械水平上引起衰老。 随着基因阵列技术的发展，已经表明，老年小鼠的组织通过改变大量基因的表达来提高应激反应并降低代谢。 已知氧化剂诱导应激反应和基因表达的改变。 在正常人二倍体成纤维细胞（HDF）中，适度剂量的氧化剂导致细胞过早地形成衰老表型。表型转换表明氧化应激后可能在分子水平上产生了多种内在变化。 p21 WAF 17/Cip 1/Sdi 1的升高被发现先于衰老表型的发作，这涉及8个衰老相关基因的表达升高。 利用微阵列技术的初步研究指出氧化剂诱导衰老相关基因以及衰老相关基因的方向。 这些观察结果使我们假设氧化剂可以诱导衰老相关基因的表达，其中一些基因由p21控制。 小鼠胚胎成纤维细胞（MEFs）将使我们能够确定可能与体内衰老相关的氧化应激的分子程序。 使用衰老表型作为MEFs中多个分子变化的标记，我们将使用涉及基因阵列和北方印迹技术的全球性和系统性方法来确定由氧化应激引起的基因表达模式。 将比较来自年轻小鼠和老年小鼠的真皮结缔组织以生成衰老基因谱。 氧化应激基因表达模式和衰老基因谱之间的比较将使我们能够严格测试氧化应激和衰老之间的关系。 这种方法也将导致识别重要的衰老相关的变化，可以研究其在细胞水平上的调节机制。由于已报道p21控制大量基因的表达，我们将使用p21敲除MEFs或HDF测试p21与氧化应激反应和皮肤结缔组织老化所共有的有限数量的功能重要基因的表达之间的关系。 最后，H2 O2脉冲处理后持续p21升高的机制将通过详细分析转录激活或mRNA稳定来确定。 由于氧化剂在我们的日常生活、衰老和疾病状态中无处不在，因此在分子水平上了解氧化剂毒性的基本机制非常重要。 我们有一个独特的发现，过早衰老与氧化应激，并将结合联合收割机在体外和体内的方法，以揭示触发不必要的影响老化。

Aging is the highest risk factor for many fatal diseases. The Free Radical Theory of Aging argues for a role of oxidant toxicity in aging. Although oxidative damage accumulates during the process of aging, it is not clear how oxidants might cause aging at the mechanistic level. As the gene array technology evolves, it has been shown that tissues from old mice elevate stress responses and decrease metabolism by altering the expression of a large number of genes. Oxidants are known to induce stress responses and alterations of gene expression. In normal human diploid fibroblasts (HDFs) mild doses of oxidants cause the cells to develop a senescent phenotype prematurely. The phenotype switch suggests that multiple intrinsic changes may have been produced at the molecular level following oxidative stress. Elevation of p21 WAF17/Cip1/Sdi1 was found to precede the onset of the senescent phenotype, which involves an elevated expression of 8 senescence-associated genes. Preliminary studies using the microarray technology point to the direction that oxidants induce aging-associated genes as well as senescence- associated genes. These observations lead us to hypothesize that oxidants can induce the expression of aging-associated genes, some of which are controlled by p21. Mouse embryonic fibroblasts (MEFs) will allow us to determine the molecular program of oxidative stress that might be relevant to aging in vivo. Using the senescent phenotype as a marker of multiple molecular changes in MEFs, we will determine the gene expression pattern resulting from oxidative stress using a global and systematic approach involving gene array and Northern blot techniques. Dermal connective tissues from young and old mice will be compared to generate an aging gene profile. Comparison between the pattern of oxidative stress gene expression and the aging gene profile will allow us to critically test the relationship between oxidative stress and aging. This approach will also lead to the identification of important aging-associated changes that can be studied for their mechanisms of regulation at the cellular level. Since p21 has been reported to control the expression of a large number of genes, we will test the relationship between p21 and the expression of a limited number of functionally important genes shared by oxidative stress response and aging of dermal connective tissue using p21 knockout MEFs or HDFs. Finally, the mechanism of sustained p21 elevation following a pulse treatment of H2O2 will be determined by detailed analysis of transcriptional activation or mRNA stabilization. Because of the ubiquitousness of oxidants in our daily life, aging and disease states, it is important to understand the fundamental mechanisms of oxidant toxicity at the molecular level. We have a unique finding of premature senescence with oxidative stress and will combine in vitro and in vivo approaches to uncover the trigger of unwanted effects of aging.