天然免疫调节分子SP-D在慢性阻塞性肺疾病中的作用机制

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and the prevalence is predicted to increase in the coming two decades. Cigarette smoking, ambient air pollution (especially high level of PM2.5) as well as some human gene polymorphisms (alleles) are risk factors to trigger COPD. Surfactant protein D (SP-D) in the lung plays a critical role in the host defense, regulation of immunity and inflammation, and pulmonary alveolar homeostasis. Recent studies demonstrate that SP-D expression and SP-D genetic variation are significantly associated with COPD and that individuals with SP-D C allele are more susceptible to COPD incidence compared to those with SP-D T allele. However, the mechanism of SP-D function in COPD is poorly understood so far. We hypothesize that SP-D plays a critical role in the pathogenesis of COPD through modulating immunity and inflammation, as well as related signaling pathways in the lung, and that human SP-D C allele has weaker ability compared to SP-D T allele. In this study we will establish one unique COPD model with three types of mice, i.e. Wild-type C57BL/6, SP-D knockout (KO), and humanized SP-D transgenic mice, and then investigate the roles of SP-D and human SP-D alleles in the pathogenesis of COPD on its regulation of immunity and inflammation, and related signaling pathways, using multiple techniques including respiratory physiology, histology, immunohistochemistry, cellular and molecular biology. Furthermore, this proposed study will elucidate functionally different mechanisms of human SP-D alleles associated with COPD. Successful completion of the proposal will generate first COPD model with humanized SP-D transgenic mice, which can provide a powerful in vivo tool for gene/allele-specific COPD drug development and gene/allele-environment interactions. The knowledge gained from this study will provide scientific basis for COPD therapy and personalized medicine.

慢性阻塞性肺疾病（COPD）是世界范围第四大疾病死亡原因。吸烟﹑空气污染（如高PM2.5雾霾）和某些基因的多态性是引起COPD的主要因素。表面活性蛋白D（SP-D）主要功能是宿主防卫，免疫和炎症调节及维持肺泡稳态。最近研究表明SP-D的表达和SP-D的基因型与COPD密切相关，携带SP-D C等位基因的人群比SP-D T人群对COPD更易感。其机制尚不清楚。我们假设SP-D通过调节肺免疫和炎症及信号途径来影响COPD发生，C等位基因比T有较弱的调节功能。本题旨在通过野生型﹑SP-D 基因敲除和人源化SP-D转基因小鼠的COPD模型，利用呼吸生理，肺组织病理，细胞和分子水平的多方面技术，明确SP-D在COPD病理的作用及对肺免疫和炎症信号途径的调节功能，阐明人类SP-D基因多态性导致COPD不同易感性的机制。此题将为防治COPD提供新的理论依据，为靶点药物研发提供首个人源化COPD动物模型。

人类SP-D基因有两个公认的基于单核苷酸多态性（SNP）rs721917的遗传变异体：SP-D T和SP-D C等位基因。人群研究显示SNP和COPD等慢性肺损伤疾病相关。但是，人类SP-D基因遗传变异体在COPD的病理过程中的作用并不清楚。NOD样受体家族包含pyrin结构域蛋白3（NLRP3）炎性小体和核因子κB（NF-κB）是天然免疫和炎症信号通路的关键调节因子，而在COPD病理过程中的作用也不明确。我们通过构建人源化转基因小鼠（无小鼠SP-D表达，只表达人类SP-D）hTG-T和hTG-C，并和野生型WT小鼠，SP-D敲除KO小鼠共同构建COPD模型（胰肽酶和脂多糖鼻腔吸入法），然后通过肺组织石蜡切片HE染色，光镜，western blot 杂交，免疫荧光，TUNEL法凋亡检测，ELISA酶联免疫吸附测定等方法进行四组小鼠肺组织的病理，细胞和分子的检测。用T-TEST和ANOVA进行统计分析。结果发现：在脂多糖加胰肽酶联合吸入4周以后，各组小鼠病理显示出同COPD类似的慢性肺损伤病理特征。在COPD组，KO小鼠，hTG-C小鼠与WT及hTG-T小鼠相比，显示出更高的肺损伤指数，支气管肺泡灌洗液中存在更多的炎性细胞，更多的凋亡活化（TUNEL，活化的Caspase-3和Caspase-8）；同时，hTG-T小鼠显示出与WT小鼠相似的肺损伤程度。hTG-C小鼠和SP-D KO小鼠肺组织中TNF-α，IL-6和IL-1β的表达水平相较于WT及hTG-T小鼠显著升高。KO和hTG-C小鼠肺组织中NLRP3炎性小体的活化水平（NLRP3，ASC，Caspase-1）和活化的NF-κB水平比WT及hTG-T显著升高。我们的研究结果显示：人类SP-D的遗传变异体（SP-D T和SP-D C）通过抑制细胞凋亡，抑制炎性小体和NF-κB活化，在COPD病理过程中起到不同的保护作用。