PDCD4通过调控巨噬细胞的替代活化介导哮喘气道重塑的分子机制研究

Asthma is characterized pathologically by structural changes in the airway, termed airway remodeling. Macrophage alternative activation influences airway remodeling to a great degree. But little is known about the underlying mechanism yet. Our previous study found that the silence of programmed cell death 4 (Pdcd4) gene down-regulated macrophage alternative activation phenotype and airway remodeling in our asthma model of E3 rats. Based on these experiments, this project is going to find the regulation molecules associated with PDCD4 regulating macrophage alternative activation with protein chip technology. After verifying the expression of these regulation molecules in NR8383 cells and rat primary bone marrow macrophages by Western blotting, ELASA, real-time quantitative PCR (RT-qPCR) and so on, the overexpression vector and the RNAi plasmid of the regulation molecules will be constructed and transfected into NR8383 cell line, respectively. Then the stably transfected cells are selected by G418 and the markers of macrophage activation, including induced nitricoxide synthase (iNOS), interleukin-12 (IL-12), found in inflammatory zone 1 (Fizz1), arginase-1(Arg-1), Ym1/2, and transforming growth factor-β1 (TGF-β1) are detected at mRNA level and protein level to confirm the role of the regulation molecules. Moreover, RNAi in vivo is performed in E3 rats through intranasally instilling with RNAi plasmid of the regulation molecules during the period of asthma model induction. The frequently-used asthmatic indices, such as lung/body-weight ratio, total cell number and different cell-type ratios in BALF, total IgE and OVA-specific IgG1 level in serum, and concentration of NO in serum are analyzed. The expressions of IL-12, Fizz1, Ym1/2, Arg-1, TGF-β1 and those regulation molecules are analyzed with RT-qPCR and Western blotting. ARG-1 activity is determined by measuring arginine-derived urea in the lung tissue extracts. And lung tissues are stained with haematoxylin eosin staining to observe pathological changes. Masson staining is performed for the detection of bronchus collagen fibers. PAS staining is performed to detect bronchus mucus secretion. The mass of hydroxyproline, which is often used to reflect the synthesis of collagen, is analyzed in lung tissue. To find the upstream regulation mechanisms, promoter function analysis, site-directed mutagenesis and Chromatin Immunoprecipitation are used to confirm the key transcription factors for Pdcd4 to modulate macrophage alternative activation. Finally, the expressions of PDCD4, the above-mentioned regulation molecules and the key transcription factors in asthma children periphery blood white cells are detected to analyze their relationships with asthma condition, thus revealing the regulatory mechanisms of PDCD4 and providing novel molecular targets for asthma treatment.

巨噬细胞的替代活化是影响支气管哮喘气道重塑的重要因素，但机制未完全明了。在前期实验发现干扰Pdcd4基因可下调大鼠肺泡巨噬细胞替代活化的表型、减轻大鼠气道重塑严重程度的基础上，本研究提出“PDCD4调控巨噬细胞的替代活化影响哮喘气道重塑”的新设想。在大鼠巨噬细胞系中上调与下调PDCD4的表达，采用蛋白芯片技术筛选出与巨噬细胞替代活化相关的调控分子，之后设计在体与离体实验观察调控分子的表达变化对巨噬细胞替代活化表型及气道重塑的影响，揭示PDCD4的下游调控机制；通过启动子活性检测、染色质免疫共沉淀等技术确定参与Pdcd4 基因表达的关键转录因子，揭示PDCD4的上游调控机制；在哮喘患儿与对照儿童外周血白细胞中验证PDCD4、调控分子及关键转录因子的表达差异，明确本研究的临床意义。旨在确定和阐明PDCD4影响巨噬细胞替代活化的作用和分子机制，为哮喘的防治及新药研发提供新的理论和实验依据。

项目的背景：程序性细胞死亡因子4（Pdcd4）参与炎症和代谢性疾病的过程，但其机制仍待研究。.主要研究内容：为探讨Pdcd4在巨噬细胞中的作用，将Pdcd4下调慢病毒转染THP1细胞。观察在游离脂肪酸（FFAs）处理的细胞模型中Pdcd4的表达和分布情况，以及Pdcd4敲除慢病毒载体和Pdcd4过表达载体改变BRL 3A细胞中Pdcd4的表达后BRL 3A细胞表型的改变。.重要结果及关键数据：1、采用RT-qPCR和western blot检测Pdcd4在THP1细胞中的表达。与NC组相比，KD组的THP1细胞表现出Pdcd4表达水平降低。RNA-seq分析显示KD组和NC组的THP1细胞中29543个基因的表达水平，其中，共测定了4932个DEGs，包括2466个上调基因和2466个下调基因。KEGG通路分析显示27条信号通路显著丰富，其中9条与DEGs上调相关，18条与DEGs下调相关。剪接体、细胞周期、真核生物中核糖体的发生、RNA转运和蛋白酶体是与上调DEGs相关的前5条富集途径。对于下调的DEG，前5个富集途径是类风湿关节炎、细胞因子-细胞因子-受体相互作用、造血细胞谱系、溶酶体和c型凝集素受体信号途径。对于20个上调的DEGs， qPCR验证显示其中9个基因的表达水平明显上调。对于10个下调的DEG，qPCR结果显示这10个基因的表达水平明显下调。.2、经FFA处理的BRL 3A细胞中Pdcd4表达增加。Pdcd4基因敲除显著抑制FFA诱导的BRL 3A细胞脂质的积聚，Pdcd4过表达加速FFA诱导的BRL 3A细胞脂质的积聚。此外，过氧化物酶体增殖物激活受体α（Pparα）敲除可减弱Pdcd4敲除细胞中酰基辅酶A氧化酶1-3（Acox1-3）的表达，从而最终将脂质沉积恢复到正常水平。.科学意义：Pdcd4参与THP1细胞和BRL 3A细胞的生物学过程，为后续机制研究奠定基础。