An approach to the pathogenesis of inflammatory lung diseases by establishing efficient analytical protocols for multiple mediator gene polymorphisms

1.趋化因子等基因多态性的鉴定和分析：首先，我们分析了日本健康对照和肺结核患者中NRAMP 1基因的各种基因多态性。在所分析的多态性中，一个和两个GT重复的罕见组合在肺纤维化（PF）患者中更常见。然后，我们分析了日本健康对照组和肺纤维化患者的纤连蛋白SNP。通过检测MCP-1启动子-2518 SNP，我们发现G等位基因在日本人中更常见，但在PF患者中的频率没有差异。在IL-8基因分析中，我们通过对IL-8基因启动子区1 Kb序列的直接测序，发现该SNP在脓毒症患者中的频率分布为1：3：6，在健康对照组中的频率分布为16：47：37（A/A：A/T：T/T）.各种介质基因在动物模型中表达的分析：（1）放射性纤维化大鼠模型：我们分析了趋化因子MCP-1、MIP-1 α、β、MIP-2、趋化因子和fractalkine的基因表达，发现MCP-1、TARC mRNA在照射后4周表达增强，MCP-1表达略有增加，2）烧伤致敏脓毒症和ARDS的小鼠模型：在无致敏组中72小时存活率为100%，在烧伤致敏模型中为0%。血浆细胞因子水平在未致敏组呈单峰型升高，在烧伤致敏组呈双峰型升高。治疗后1周，烧伤致敏组肝脏IL-18含量显著高于假手术组。

1. Identifiticantion and analysis of chemokine, etc gene polymorphisms: Initially we analyzed various gene polymorphisms of a NRAMP1 gene in Japanese healthy controls and patients with pulmonary tuberculosis. Among polymorphisms analyzed, the rare combination of one and two GT repeats was more frequent in patients with pulmonary fibrosis (PF).We then analyzed fibronectin SNPs in Japanese healthy controls and patients with pulmonary fibrosis. Although these frequency was quite different between Japanese and Caucasians, it was not different in PF patients.By examining MCP-1 promoter -2518 SNP, we found that allele G was more frequent in Japanese, but its frequency was not different in PF patients. Plasma MCP-1 levels was not different among MCP-1 genotypes.For IL-8 gene analysis, we searched new polymorphisms by directly sequencing 1 Kb promoter region and found that the frequency distribution of this SNP was 1:3:6 in a sepsis patient and 16:47:37 (A/A:A/T:T/T) in healthy controls.2. Analysis of various mediator genes expression in animal models: (1) A rat model of radiation fibrosis: We analyzed the gene expressions of chemokines, MCP-1, MIP-1alpha, beta, MIP-2, lymphotactin, and fractalkine and found the augmented expression of MCP-1, TARC mRNA at four weeks after irradiation, and a slightly increased expression of MCP-1, MIP-1alpha at 8 weeks.2) A mouse model of burn-primed sepsis and ARDS:The 72 hour survival was 100% in no priming group and 0% in a bum primed model. The plasma cytokine increase was single-peaked in no priming group and two-peaked in burn-primed group. Furthermore, liver IL-18 content was significantly higher in burn-primed group than in sham group one week after the treatment.