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

将Klotho cDNA插入pCAGGS载体中制备Klotho表达质粒，转染细胞后经western blotting证实Klotho蛋白的产生。我们用从COS-1细胞中提取的培养液培养HUVEC细胞，其中处理了Klotho质粒。结果表明，Klotho具有抗氧化作用，SOD活性被激活，MnSOD表达增强。为了证实这种抗氧化作用，我们将klotho质粒注入小鼠尾静脉。肝脏和肾脏均产生Klotho蛋白，肝脏和肾脏均发现SOD活性被激活，LPO浓度降低。我们研究了Klotho蛋白对血压的影响。将Klotho质粒输注到高血压、SHR和SHR- sp动物模型中，分别于6周、12周后测血压，均未见对血压有影响。尽管如此，SOD活性被激活，LPO浓度降低，与正常小鼠相同。而给予NO合成酶抑制剂L-NAME后，其抗氧化作用减弱，说明Klotho的抗氧化作用部分是通过NO途径实现的。我们用含有Klotho蛋白的培养液孵育不同细胞，测定细胞内cAMP浓度和PKC活性，探讨Klotho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脏细胞中升高。结果表明，Klotho基因表达不同，其信号通路也不同。将Klotho质粒插入COS-1细胞后，可抑制1 α - oase、维生素D活化酶等基因的表达，揭示了Klotho与钙代谢的关系。

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.