HYDROXYL RADICAL BIOLOGY BY CEREBRAL ISCHEMIA

目前，这个国家有200多万公民是 因中风而禁用。由脑缺血引起的中风是第三种 导致死亡的主要原因和导致神经残疾的主要原因。 每年因中风造成的直接和间接损失估计为25美元。 十亿美元。功能部分恢复常见于卒中患者 病人。然而，功能恢复的分子机制是 人们对此知之甚少。在一个发育良好的啮齿动物中风模型中， 脑缺血会导致超氧化物和过氧化氢的增加， 它们被转化为羟基自由基。羟基自由基，一种已知的 诱变剂诱导DNA修复合成及修复酶表达 细胞培养中的DNA聚合酶-β。因此，羟基自由基能 引起导致迟发性神经元死亡的遗传程序的激活 (细胞凋亡)。减少大脑中的细胞凋亡是发挥功能的核心 中风后的康复。 该假说认为，细胞凋亡是通过DNA损伤介导的。 脑缺血期间和脑缺血后的氧自由基。初步 脑局灶性卒中模型的实验室研究 缺血30分钟后，S1核酸酶敏感性消失 DNA聚合酶-βmRNA在细胞内的表达 缺血2小时后，脑内基因突变频率增加6倍 大蓝转基因小鼠LACL基因8小时及DNA的出现 细胞碎裂是细胞凋亡的一个特征，在缺血后3天。它 从别人和我们的研究中得知，基因突变是 防止在DNA修复合成过程中引入的错误。突变可能导致 导致神经系统因缺血而发生细胞凋亡。的目标是 这项建议是为了确定羟基自由基对 脑缺血后脑基因突变与细胞凋亡。在这 建议，我们将： (1)分析150个Lacl突变体的DNA序列变化。 缺血组和30例正常脑组织LACL突变组； (2)测定神经营养素链的DNA修复 聚合酶-β、p53和肌动蛋白基因； (3)测量DNA中8-羟基脱氧鸟苷(Oh8dG)的增加。 缺血后皮质细胞； (4)确定两种疾病的形成是否存在正相关 ···与生理参数的增加 静脉注射突变频率、DNA修复、oh8dG和细胞凋亡 缺血前输注超氧化物歧化酶和过氧化氢酶。 从这个项目中获得的突变谱将是一个直接的 脑缺血后羟基自由基致DNA损伤的指标。这 该模型适用于氧的病理生物学效应的研究 压力，以及测试减少或消除毒副作用的药物 中风后的羟基自由基。

Currently there are more than 2 million citizens in this country who are disabled by stroke. Stroke caused by cerebral ischemia is the third leading cause of death and the leading cause of neurological disability. The annual direct and indirect loss from stroke is estimated to be $25 billion. Partial recovery of function is frequently noted in stroke patients. The molecular mechanism of functional recovery, however, is poorly understood. In a well developed stroke model of the rodents, cerebral ischemia causes an increase in superoxide and hydrogen peroxide, which are converted to hydroxyl radicals. Hydroxyl radical, a known mutagen, induces DNA repair synthesis and the expression of repair enzyme DNA polymerase-beta in cell culture. Therefore, hydroxyl radical could cause activation of a genetic program leading to delayed neuronal death (apoptosis). To reduce apoptosis in the brain is central to functional recovery after stroke. The hypothesis is that apoptosis is mediated via DNA damage by the elevated oxygen free radicals during and after cerebral ischemia. Preliminary studies from our laboratory using th stroke model of focal cerebral ischemia of 30 min demonstrated a disappearance of S1 nuclease sensitive sites and an increase in the expression of DNA polymerase-beta mRNA within 2 hr after ischemia, a 6-fold elevation in gene mutation frequency within 8 hr in the lacl gene of Big Blue transgenic mice, and an appearance of DNA fragmentation, a characteristics of apoptosis, 3 days after ischemia. It is known from studies by others and ours that gene mutations are results from errors introduced during DNA repair synthesis. Mutations could lead to apoptosis in the nervous system as a results of ischemia. The goal of this proposal is to determine the contribution of hydroxy radicals to cerebral gene mutation and apoptosis after cerebral ischemia. In this proposal, we will: (1) analyze the sequence change in the DNA of 150 lacl mutants from ischemia and 30 lacl mutants from normal brain samples; (2) determine DNA repair in the transcribed strand of neurotrophin, DNA polymerase-beta, p53 and actin genes; (3) measure the increase in 8-hydroxyldeoxyguanosine (oh8dG) in DNA from cortical cells after ischemia; (4) determine if there is a positive correlation between the formation of hydroxyl radicals and the increase in the physiological parameter of mutation frequency, DNA repair, oh8dG and apoptosis using intravenous infusion of superoxide dismutase plus catalase before ischemia. The mutation spectrum that is obtained from this project will be a direct indicator of DNA damage by hydroxyl radicals after cerebral ischemia. This model will be suitable for exploring pathobiological effect of oxygen stress, and for testing drugs that reduce or abolish the damaging effect of hydroxyl radical after stroke.