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Functional analysis of senescence related Klotho gene and its application for the therapy

衰老相关Klotho基因的功能分析及其在治疗中的应用

基本信息

批准号：
15590342
负责人：
RAKUGI Hiromi
金额：
$ 2.18万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590342/
关键词：
Klotho SOD oxidative stress NO blood pressure 老化ビタミンD Ca代謝 PKC 血管老化 MnSOD 一酸化窒素内皮細胞 cAMP 血管

项目摘要

Klotho expression plasmid was produced by inserting Klotho cDNA into pCAGGS vector, and the production of Klotho protein was confirmed by western blotting after transfection into the cells. We incubated HUVEC cells with the medium taken from COS-1 cells into which Klotho plasmid was transacted., and it was revealed that Klotho has anti-oxidative effect from the result that SOD activity was activated, and the expression of MnSOD was enhainced. To confirm this anti-oxidative effect in vivo, we infused klotho plasmid into mouse tail vein. Klotho protein was produced in Liver and kidney, and it was found that SOD activity was activated, LPO concentration was reduced in both Liver and kidney.We investigated the effect of Klotho protein on blood pressure. Klotho plasmid was infused into the animal model of hypertension, SHR and SHR-SP, and the blood pressure was measured after 6weeks, 12 weeks, however, no effect was found on blood pressure. In spite of that, SOD activity was activated, and LPO concentration was reduced as same as normal mouse. Moreover this anti-oxidative effect was reduced when L-NAME, inhibitor of NO synthase, was administrated which shows that anti-oxidative effect by Klotho is partly through the NO pathway.We measured intracellular cAMP concentration and PKC activity by incubating with medium containing Klotho protein on various cells to investigate the signaling pathway of Klotho. The result was that cAMP concentration was high in most cells, however, PKC activity was only increased in testis and kidney cells where Klotho gene is expressed. It was revealed that Klotho signaling pathways are different depends on its gene expression.1 alpha-OHase, vitamin D activating enzyme, gene expression was suppressed when Klotho plasmid was inserted into COS-1 cell, which shower the relation of Klotho with Ca metabolism.

通过将Klotho cDNA插入pCAGGS载体中制备Klotho表达质粒，并在转染细胞后通过Western印迹证实Klotho蛋白的产生。我们将HUVEC细胞与取自COS-1细胞的培养基一起孵育，Klotho质粒被转入该培养基中。Klotho能激活SOD活性，增强MnSOD的表达，表明Klotho具有抗氧化作用。为了证实这种体内抗氧化作用，我们将klotho质粒注入小鼠尾静脉。Klotho蛋白在肝脏和肾脏中产生，并能激活肝脏和肾脏中的SOD活性，降低肝脏和肾脏中的LPO含量，研究Klotho蛋白对血压的影响。将Klotho质粒分别注入高血压动物模型、SHR和SHR-SP，6周、12周后测血压，但对血压无影响。尽管如此，SOD活性仍被激活，LPO浓度降低，与正常小鼠相同。本研究通过测定细胞内cAMP浓度和PKC活性，探讨Klotho的抗氧化作用的信号通路。结果表明，在大多数细胞中cAMP浓度较高，而PKC活性仅在Klotho基因表达的睾丸和肾细胞中升高。结果表明，Klotho的信号通路因其基因表达不同而不同，Klotho质粒导入COS-1细胞后，维生素D激活酶1 α-OHase基因表达受到抑制，表明Klotho与钙代谢有关。

项目成果

期刊论文数量（54）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

G2736A polymorphism of thiazide-sensitive Na-Cl cotransporter gene predisposes to hypertension in young women

DOI：
10.1097/00004872-200411000-00014
发表时间：
2004-11
期刊：
Journal of Hypertension
影响因子：
4.9
作者：
A. Matsuo;T. Katsuya;K. Ishikawa;K. Sugimoto;Y. Iwashima;Koichi Yamamoto;M. Ohishi;H. Rakugi;T. Ogihara
通讯作者：
A. Matsuo;T. Katsuya;K. Ishikawa;K. Sugimoto;Y. Iwashima;Koichi Yamamoto;M. Ohishi;H. Rakugi;T. Ogihara

Adiponectin I164T mutation is associated with the metabolic syndrome and coronary artery disease