一种新颖的测定和分析羟自由基氧化损伤RNA及其分子机理的化学发光体系

A Novel Chemiluminescence System for Detecting and Analyzing Oxidative Damaged RNA by Hydroxyl Radical and Its Molecular Mechanism

近年来 ,羟自由基 ( ·OH)对DNA氧化损伤已受到广泛关注。但是很少研究·OH对RNA的氧化损伤。其实 ,RNA与DNA一样 ,也是核酸的两大组分之一 ,也有许多重要功能。所以·OH攻击RNA也会引起严重后果 ,会造成细胞功能衰退甚至细胞死亡等。为此 ,我们建立了Vit C CuSO4 Phen H2 O2 RNA这一产生和测定·OH氧化损伤RNA的化学发光体系 ,以便加强·OH氧化损伤RNA的研究。通过对本体系测定条件的研究 ,得出了本体系最佳组方是 :Vit C、CuSO4 、Phen、H2 O2 和RNA ,浓度分别为 35 0 μmol/L、5 5 μmol/L、35 0 μmol/L、0 2mol/L和 2 0 μg/mL ,体系pH为 5 5 ,体系终体积为 1mL。随后 ,利用本体系检测了槲皮素、咖啡酸、黄芩甙和芦丁抗·OH氧化损伤RNA的作用 ,发现这四种抗氧化剂均能有效抑制·OH对RNA的氧化损伤 ,从而也证明了本体系的可行性和实用性。我们也利用此体系和特异抗氧化剂分析了·OH氧化损伤RNA的分子机理 ,结果发现 ,·OH清除剂硫脲几乎抑制全部发光 ,推测是因硫脲清除了引发剂·OH而不能引发RNA氧化损伤所致 ;过氧化氢酶几乎能抑制全部发光 ,推测是因过氧化氢酶清除了H2 O2 而不能产生引发剂·OH所致 ;O2 ·清除剂SOD只能抑制小部分发光 ;1O2 清除剂叠氮化钠和苯甲酸都能抑制绝大部分发光。这?

In recent years, oxidative damaged DNA by hydroxyl radical ( ·OH) has been received much attention. But the reports on oxidative damaged RNA by ·OH were quite limited. In fact, RNA, being the same as DNA, is also one of big two components for nucleic acid. It also has many important functions. Therefore, RNA attacked by ·OH leads to serious consequence, which bring about decline of the cell activity even death of the cell. Thus a chemiluminescence system for generating and detecting oxidative damaged RNA by ·OH was made, which initiated with Vit.C/CuSO 4/Phen/H 2O 2/RNA, so as to strengthen the study on oxidative damaged RNA by ·OH. Through the investigation on detecting conditions of the system, optimal scheme of the system was obtained: concentration of Vit.C, CuSO, Phen, H 2O 2 and RNA were 350μmol/L,55μmol/L,350μmol/L,0.2mol/L and 20μg/mL,respectively. The pH of the system was 5.5. The final volume was 1mL. Using the system, we detected the effects that quercetin, caffeic acid, baicalin and rutin resisted oxidative damaged RNA by ·OH and found that the four antioxidants could effectively inhibited oxidative damaged RNA by ·OH. These facts also proved feasibility and practicality of the system. Molecular mechanism for oxidative damaged RNA by ·OH was analyzed by the system and special antioxidants. The results indicated that sulfocarbamide which was a ·OH scavenger nearly inhibited whole chemiluminescence. The reason for the phenomenon was that RNA oxidative damage was not initiated only because sulfocarbamide scavenged ·OH , an initiator; catalase also inhibited whole chemiluminescence, revealing that ·OH couldn't be generated due to H 2O 2 removed by catalase. SOD, a O 2 · scavenger, could only inhibit a small part of chemiluminescence, whereas NaN 3 or benzene carboxylic acid, a 1O 2 scavenger, could inhibit most of chemiluminescence. These results suggested that ·OH is an initator; but O 2 · is a minor factor, 1O 2 is a major factor for the RNA oxidative damage.