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

1.纯合突变klotho小鼠（KL-/-）肺气肿分析。(1) KL-/和野生型小鼠（WT）肺组织基因表达谱的比较。缺乏klotho基因表达的KL-/-在4周龄时发生肺气肿。我们通过Atlas Nylon膜杂交方法比较了2周龄KL-/和WT在肺中的基因表达谱。与WT相比，KL-/-肺中caspase 3、与Notch信号转导通路相关的自由基边缘同源前体、激活素受体IIA、IIB和与tgf - β家族蛋白相关的MAD同源基因7的表达更强烈。另一方面，丝氨酸蛋白酶抑制剂2基因在KL-/-肺中的表达强度较低。这些发现表明，与肺发育和肺构成细胞凋亡相关的基因的紊乱表达参与了klotho小鼠肺气肿的发病机制。蛋白酶抑制剂活性的抑制，保护肺免受各种损伤，是另一个促成因素。(2) KL-/-治疗肺气肿的研究。在雄性KL-/-中，饲粮限磷可上调klotho基因的表达。我们研究了klotho基因的上调是否能改善KL-/-中的肺气肿。3周龄（幼龄组）和5周龄（老龄组）分别饲喂限磷（0.4%-P）饲粮2周和4周。青壮年组肺未发生肺气肿，老年组肺出现严重肺气肿。klotho基因产物的补充对肺气肿的治疗有潜在的作用。人klotho基因PCR测序系统的建立。设计引物组用于PCR测定人klotho基因的DNA序列。

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.