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起着作用。虽然氧化损伤在衰老过程中积累，但氧化剂如何在机制水平上导致衰老尚不清楚。随着基因阵列技术的发展，研究表明，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，降低新陈代谢。众所周知，氧化剂可以诱导应激反应和基因表达的改变。在正常人的二倍体成纤维细胞(HDF)中，温和剂量的氧化剂会导致细胞过早地出现衰老表型。这种表型转换表明，氧化应激后可能在分子水平上产生了多种内在变化。P21WAF17/Cip1/Sdi1的升高被发现先于衰老表型的开始，这涉及到8个衰老相关基因的表达上调。使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(MEF)将使我们能够确定氧化应激的分子程序，这可能与体内衰老有关。我们将利用衰老表型作为MEF多个分子变化的标志，利用基因阵列和Northern印迹技术，通过全球和系统的方法来确定氧化应激导致的基因表达模式。来自年轻和老年小鼠的真皮结缔组织将被比较，以产生衰老基因图谱。氧化应激基因表达模式和衰老基因图谱的比较将使我们能够批判性地测试氧化应激与衰老之间的关系。这一方法还将导致识别与衰老相关的重要变化，这些变化可以在细胞水平上研究其调节机制。由于p21已经被报道控制大量基因的表达，我们将使用p21基因敲除的MEF或HDFS来测试p21与氧化应激反应和真皮结缔组织衰老所共有的有限功能重要基因的表达之间的关系。最后，脉冲处理过氧化氢后p21持续升高的机制将通过详细的转录激活或mRNA稳定分析来确定。由于氧化剂在我们的日常生活、衰老和疾病状态中普遍存在，从分子水平上了解氧化剂毒性的基本机制是非常重要的。我们有一个独特的发现，即过早衰老与氧化应激有关，并将结合体外和体内的方法来揭示衰老有害影响的触发因素。

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.