急性肺损伤中颗粒蛋白前体的microRNA调控机制研究

Progranulin ( PGRN ) is a multifunctional regulatory protein. In our previous study we found that PGRN can obviously improve the acute lung injury (ALI), but the role and mechanism is still unclear. We found that PGRN can inhibit the inflammation and improve the prognosis of ALI induced by LPS on our previous study, but the PGRN microRNAs ( miRNAs ) regulatory mechanisms are unclear. On this project we will evaluated the fluctuations of PGRN and miRNAs expression profile in LPS irritated neutrophils firstly. To explore the mechanism of PGRN regulation for neutrophil inflammation, we will also define the effect of PGRN to inflammatory signaling pathways including NF-κ B and downstream cytokines like TNF-α、IL-10 and IL-1βthrough PGRN overexpression and SiRNA interference. According to the target gene sequence from miRNA database we will select one or more miRNA for farther study. Through cell transfection with miRNA minics and antagonist,we will to explore the regulation of miRNAs on PGRN and inflammatory release function on LPS stimulated neutrophils. Followed by lentiviral vector transfection technique, miRNA antisense nucleotide sequence ( ASO ) particles will be constructed. Both PGRN and miRNA ASO will be spray into mice body with ALI induced by LPS to assess the effects of PGRN and miRNA ASO on the inflammation response and the prognosis during ALI. The project will provide experimental data for clarifying the mechanism of PGRN against inflammation and regulation which controled and regulated by miRNAs.

颗粒蛋白前体（PGRN）是多功能调节蛋白，其在急性肺损伤（ALI）中的作用和机制尚有待于阐明。我们前期研究发现PGRN可以抑制炎症并改善ALI的预后，但PGRN 的microRNAs（miRNAs）调控机制尚有待于研究。本课题拟首先通过中性粒细胞培养探讨LPS刺激下PGRN和miRNAs表达变化。然后，应用PGRN过表达和SiRNA干扰方法探讨PGRN对中性粒细胞炎症信号通路和下游炎症因子的影响。通过miRNA数据库分析并筛选对PGRN可能起调控作用的miRNA。通过细胞转染miRNA minics和拮抗剂方法探讨miRNAs对中性粒细胞中PGRN的调控及炎症释放的作用。在此基础上，运用慢病毒载体转染技术，构建miRNA 反义核苷酸序列（ASO）颗粒，明确miRNA ASO对ALI中PGRN、炎症反应和肺损伤预后的影响，为阐明PGRN在ALI中的作用和miRNA调控机制提供实验依据。

急性肺损伤（ALI）/急性呼吸窘迫综合征是一种常见的临床危重症，发病率和死亡率极高。难以控制的炎症反应和肺泡上皮细胞凋亡在ALI发生发展过程中发挥重要作用。我们前期研究发现颗粒蛋白前体（progranulin, PGRN）可以缓解ALI严重程度，然而PGRN在调控急性肺损伤时肺部炎症和肺泡上皮细胞凋亡中作用以及PGRN的miRNA调控机制尚有待于研究。我们采用小鼠急性肺损伤模型，探索ALI不同时间点肺组织中PGRN表达的变化特征，通过过表达/低表达肺组织中PGRN，探讨PGRN在ALI发生发展中的作用，利用生物信息学方法筛选潜在调控PGRN的miRNA并检测这些miRNA在ALI前后肺组织中的表达差异，利用细胞功能学实验和在体实验验证特异性调控PGRN的miRNA在ALI发病中调控肺部炎症反应和肺泡上皮细胞凋亡中的作用。研究结果表明抑制体内miRNA-34b-5p表达通过作用PGRN缓解肺部炎症反应和肺泡上皮细胞凋亡进而减轻ALI严重程度。颗粒蛋白前体是一种内源性抗炎蛋白，本项目研究了颗粒蛋白前体在急性肺损伤中变化特征及功能，进一步明确了其在体内的调控方式，为急性肺损伤治疗提供了新的靶点。研究结果具有一定的理论意义和现实意义。