锌指蛋白A20对肺泡巨噬细胞炎症反应活性的调控在急性肺损伤中的作用及机制研究

Excessive inflammatory response is the key pathophysiological characteristics of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). A20 protein regulates the inflamotory response of a variety of inflammatory diseases, and our preliminary study found that A20 expression increased in LPS-induced ALI in vivo and alveolar macrophages stimulated by LPS in vitro, but its role in ALI remains unclear. The project intends to stimulate alveolar macrophages by LPS in vitro, design A20 expression and silencing vector, transfect macrophages, and to study its influences on cytokine secretion; use inhibitors to block the key enzymes of the inflammatory signal pathways,such as MAPK and NF-κB, and then to detect the expression of critical protein of inflammatory response, as to explore the mechanism of A20 on the cytokine secretion of alveolar macrophage; establish ALI model caused by cecal ligation and puncture-induced sepsis, prepare A20 gene and anti-CD68 (macrophage-specific antigen) antibody conjugated immunoliposome, transfect rats, assess the impact of A20 on the inflammatory response of ALI,as to clarify the role and mechanism of A20 on the regulation of inflammation in ALI. This study will further reveal the mechanisms of inflammatory regulation in ALI, and provide some theoretical and experimental basis for clinical treatment of ALI.

过度放大的炎症反应是急性肺损伤（ALI）/急性呼吸窘迫综合征的关键病理生理特征。A20蛋白调控机体多种炎症性疾病的炎症反应，我们前期研究发现A20在LPS诱导ALI及LPS刺激后的肺泡巨噬细胞中表达增加，但其在ALI中的作用及机制尚不清楚。本项目拟通过体外LPS刺激肺泡巨噬细胞，慢病毒构建A20真核表达及沉默载体，转染巨噬细胞，研究其对巨噬细胞细胞因子分泌的影响；并分别阻断MAPK、NF-κB等炎症相关信号通路中的关键酶，检测相关信号活化及关键蛋白表达变化，探索A20抑制肺泡巨噬细胞细胞因子分泌的作用机制；体内复制大鼠盲肠结扎穿孔所致脓毒血症ALI模型，制备A20基因与抗CD68（巨噬细胞特异性抗原）抗体偶联的免疫脂质体，体内转染，评估A20对ALI炎症反应的影响，阐明A20对ALI炎症调控的作用及机制。本研究将进一步揭示ALI炎症调控机制，为临床ALI的治疗提供部分理论依据及实验基础。

控制过度放大的炎症反应对急性呼吸窘迫综合征至关重要。A20 蛋白是一种泛素化修饰酶，能够调控机体多种炎症反应，且主要通过NF-κB信号通路。本项目主要研究A20对肺泡巨噬细胞炎症反应的调控在急性肺损伤中的作用及机制。构建静脉注射LPS所致急性肺损伤模型，收集各个时间点肺泡灌洗液（BALF）及肺组织，ELISA法测定BALF中IL-1β、TNF-α水平及肺组织中NF-κB活性，Western Blot及RT-PCR法测定A20的蛋白及mRNA。双重免疫荧光标记法检测A20在肺泡巨噬细胞中的表达情况。慢病毒构建 A20 真核表达及沉默载体，转染肺泡巨噬细胞，并用LPS进行干预，收集各个时间点的培养上清液和肺泡巨噬细胞。ELISA法和RT-PCR法分别测定培养上清液和肺泡巨噬细胞中IL-1β及TNF-α水平；ELISA法测定肺组织中NF-κB活性，Western Blot测定NF-κB P65蛋白表达，Western Blot及RT-PCR法测定A20的蛋白及mRNA。采用流式细胞技术检测A20对肺泡巨噬细胞分型（M1和M2）的影响。结果显示LPS所致急性肺损伤时，肺组织中A20、TNF-α、IL-1β及NF-κB活性都升高，且高峰在2h。急性肺损伤早期，A20在肺泡巨噬细胞中大量表达。体外研究中，A20能够抑制肺泡巨噬细胞分泌及表达TNF-α、IL-1β，并使NF-κB蛋白表达及活性都降低。A20能够促进M1型肺泡巨噬细胞向M2型转化。综上，A20可通过NF-κB及促进M1型肺泡巨噬细胞向M2转化，调控肺泡巨噬细胞炎症反应，进而减轻LPS所致肺损伤。该研究为治疗肺损伤炎症反应提供理论基础。