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

将Klotho基因插入到pCAGGS载体中，构建Klotho表达载体，转染细胞后用Western blotting检测Klotho蛋白的表达。用Klotho载体导入的COS-1细胞的培养液与HUVEC细胞孵育，结果表明Klotho具有抗氧化作用，其结果表明，Klotho对HUVEC细胞的SOD活性有激活作用，对MnSOD的表达也有增强作用。为了在体内证实这种抗氧化作用，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，发现肝脏和肾脏中的SOD活性被激活，LPO含量降低。将Klotho质粒注入高血压、SHR和SHR-SP动物模型中，分别于6周、12周测量血压，但对血压无影响。尽管如此，超氧化物歧化酶活性被激活，脂质过氧化产物含量降低，与正常小鼠相同。此外，给予一氧化氮合酶抑制剂L-NAME后，细胞内cAMP浓度和蛋白激酶C活性明显降低，说明氯乐高的抗氧化作用部分是通过NO途径实现的。结果表明，大多数细胞内cAMP浓度较高，而PKC活性仅在表达Klotho基因的睾丸和肾脏细胞中升高。研究表明，Klotho信号通路的不同取决于其基因表达的不同。1、Klotho基因插入COS-1细胞后，维生素D活化酶α-OHase基因表达受到抑制，提示Klotho与钙代谢有关。

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.