Identification of effetor molecules for reactive oxygen species and its biological significance

近年来的研究表明，ROS参与了辐射损伤、肿瘤发生、缺血再灌注损伤、糖尿病和神经退行性疾病等多种生物学现象。在这个项目中，我们建立了在石蜡包埋标本中定位ROS引起的损伤的方法。这归功于成功地产生了针对ROS诱导的损伤的共价修饰结构的抗体。表位包括8-羟基-2‘-脱氧鸟苷和4-羟基-2-壬醛修饰的蛋白质。将该技术应用于糖尿病、再灌注损伤、砷中毒、结肠癌、病毒性肝炎的动物模型以及氮三醋酸铁(Fe-NTA)诱导的大鼠氧化应激致癌模型。然后，通过对F1杂交大鼠的遗传分析，回答了氧化应激诱导的致癌过程中是否存在靶基因的问题。本研究通过对大鼠5号和8号染色体的PCR微卫星分析，发现其5号和8号染色体的杂合性丢失发生率较高，从而得出结论：p15/p16抑癌基因是氧化应激致癌模型中的主要靶基因之一。

Recent studies have clarified that reactive oxygen species(ROS)are involved in a diversity of biological phenomena including radiation damage, carcinogenesis, ischemia-reperfusion injury, diabetes mellitus and neurodegenerative diseases. In this project we have established methods to localize ROS-induced damage in paraffin-embedded specimens. This owes to a successful production of antibodies against covalently modified structures specific for ROS-induced damage. The epitopes include 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins. This technique was applied to animal models of human specimens of diabetes mellitus, reperfusion injury, arsenic intoxication, colon cancer, viral hepatitis as well as oxidative stress-induced cancer model of rats using ferric nitrilotriacetate(Fe-NTA). Then, we have answered a question whether there are target genes in the oxidative stress-induced carcinogenesis by genetic analysis using F1 hybrid rats. This study revealed that chromosome 5 and 8 of rats showed a high incidence of loss of heterozygosity with PCR microsatellite analyses, which finally lead to a conclusion that p15/p16 tumor suppressor gene is one of the major target genes in this oxidative stress-induced carcinogenesis model.