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The Observation of the cells expressing superoxide dismutase mRNA by in situ hybridization.

原位杂交观察细胞表达超氧化物歧化酶mRNA。

基本信息

批准号：
02670011
负责人：
SASAKI Junzo
金额：
$ 0.96万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670011/
关键词：
Insitu Hybridization SOD Superoxide RNA Probe Actin Digoxigemin ジゴキシゲニン

项目摘要

Recent studies show that there is a close relationship between reactive oxygen species and man diseases.In the first year, we synthesized ^<35>S-labeled RNA probe from cDNA of human Cu/Zn-type superoxide dismutase(SOD), which degrades reactive oxygen species and investigated the expression of SOD in normal rat tissues by in situ hybridization(ISH). In addition, ISH using nonradioactive probes was examined. As a resuly, the signals showing the presence of SOD mRNA were found in hippocampus, Purkinje's cells incerebellum, hepatocytes and epithelial cells of the bronchioles. Eosinophils also showed strong signals. This is interesting finding because, eosinophils are potent producer of active oxygen species. Nonradioactive RNA probes were synthesized in the presence of digoxigenin(DIG)-UTP and were visualized with anti-DIG-alkaline phosphatase conjugate and alkaline phosphatase reaction. Weak signals of SOD mRNA were detected in the isolated eosinophils while the sigals of actin mRNA were detected under electron microscopy by nonradioactive ISH.In the last year, the expression of SOD mRNA was examined under pathological consitions. First, we tested the effect of a SOD derivative(SM-SOD)on liver injury caused by ischemia and repurfusion under electron microscopy. Postischemic reperfusion caused a drastic change in liver morphology, and intravenous administration of SM-SOD completely inhibited these morphological changes. But, the expression of endogeneous SOD in hepatocytes under these conditions was not marked. Secondly, we tested the exppession of SOD in rat lung after prolonged oxygen inhalation which caused lethal lung edema. The expression of SOD is not marked. It may be necessary to study the change of the expression of Mn type SOD.

近年来的研究表明活性氧与人类疾病有着密切的关系，本研究首先<35>从人铜锌型超氧化物歧化酶（Cu/Zn type superoxidedismutase，SOD）的cDNA中合成了15 S标记的RNA探针，并利用原位杂交技术研究了SOD在正常大鼠组织中的表达。此外，ISH使用非放射性探针进行了检查。结果表明，海马、小脑浦肯野细胞、肝细胞和细支气管上皮细胞中均有SOD mRNA的表达。嗜酸性粒细胞也显示出强烈的信号。这是一个有趣的发现，因为嗜酸性粒细胞是活性氧的有效生产者。在地高辛（DIG）-UTP存在下合成非放射性RNA探针，并用抗DIG-碱性磷酸酶缀合物和碱性磷酸酶反应显色。非放射性原位杂交（ISH）在电镜下观察到SOD mRNA的弱信号，电镜下观察到肌动蛋白mRNA的弱信号，并在病理条件下观察SOD mRNA的表达。首先，我们在电子显微镜下检测了SOD衍生物（SM-SOD）对缺血再灌注引起的肝损伤的影响。缺血后再灌注引起肝脏形态学的急剧变化，静脉注射SM-SOD完全抑制这些形态学变化。但在此条件下肝细胞内源性SOD的表达不明显。第二，观察大鼠长时间吸氧致肺水肿后肺组织中SOD的表达。SOD的表达不明显。因此，有必要研究Mn型SOD表达的变化。