Investigation of molecular mechanism in the development of chronic obstructive pulmonary disease (COPD) using transgenic animal models.

1.纯合突变型klotho小鼠（KL-/-）中肺气肿的分析。（1）KL-/-和野生型小鼠（WT）之间肺中基因表达谱的比较。缺乏klotho基因表达的KL-/-在4周龄时发生肺气肿。我们通过使用Atlas尼龙膜的杂交方法比较了2周龄时KL-/-和WT之间肺中的基因表达谱。与WT肺相比，KL-/-肺中表达的基因包括caspase 3、与Notch信号转导途径相关的自由基边缘同源物前体、激活素受体IIA、IIB和与TGF-β家族蛋白相关的MAD同源物7。另一方面，丝氨酸蛋白酶抑制剂2基因在KL-/-肺中表达较弱。这些结果表明，与肺发育和肺构成细胞凋亡相关的基因表达紊乱参与了klotho小鼠肺气肿的发病机制。蛋白酶抑制剂活性的抑制是其另一个促成因素，其保护肺免受各种损伤。（2）KL-/-肺气肿的治疗研究在KL-/-雄性中，限磷可上调klotho基因的表达。我们研究了klotho基因的上调是否改善KL-/-中的肺气肿。3周龄KL-/-组（青年组）和5周龄KL-/-组（老年组）分别饲喂限磷（0.4%-P）饲料2周和4周。青年组无肺气肿发生，老年组肺气肿严重。2.人klotho基因PCR测序体系的建立：设计PCR引物，对人klotho基因的DNA序列进行测定。

1.Analysis of the pulmonary emphysema in homozygous mutant klotho mouse (KL-/-).(1)Comparison of the gene-expression profile in the lung between KL-/-and wild-type mouse (WT).KL-/-, which lacks the klotho gene expression, develops emphysema at 4 weeks of age. We compared the gene-expression profile in the lung between KL-/-and WT at 2 weeks of age through hybridization method using Atlas Nylon Membranes. The genes including caspase 3, radical fringe homolog precursor related to a Notch signal transduction pathway, activin receptor IIA, IIB, and MAD homolog 7 related to TGF-beta family protein, were expressed more intensively in KL-/-lung than in WT lung. On the other hand, serine protease inhibitor 2 gene expressed less intensively in KL-/-lung. These findings suggest that deranged expression of the genes associated with lung development and apoptosis of pulmonary constituting cells participates in the pathogenesis of emphysema in klotho mice. The suppression of protease inhibitor activity, which protects lung from various injuries, is another contributing factor of it.(2)Investigation of therapy for emphysema In KL-/-.In male KL-/-, dietary phosphorus restriction upregulates the klotho gene expression. We investigated whether the upregulation of the klotho gene ameliorates emphysema in KL-/-. KL-/-at 3 weeks (younger group) and 5 weeks (elder group) were fed with phosphorus restricted (0.4%-P) diet for 2 and 4 weeks, respectively. The lungs of younger group did not develop emphysema while those of elder group showed severe emphysema. The replenishment of the klotho gene product is potentially useful in treating emphysema.2.Establishment of PCR system for sequencing the human klotho gene.The primer sets were designed for PCR in order to determine the DNA sequences of the human klotho gene.