NF-κB炎症信号通路靶向封闭防治急性创伤性肺损伤的实验研究

发布时间：2018-06-04 22:28

本文选题：急性肺损伤 + 核因子κB　；参考：《第四军医大学》2013年博士论文

【摘要】：背景：国内外关于ALI/ARDS的研究大多数集中于探讨细胞因子网络的失衡，尽管使用各种药物针对细胞因子进行了大量的临床防治，，但始终没有一种完全有效、可重复、有统计学意义的方法。ALI是一种炎症免疫信号转导紊乱引起的疾患。当机体受到创伤、休克等损伤时，肺是首先被攻击的靶器官和细菌来源之一。而肺泡巨噬细胞是肺组织炎症瀑布效应的激活点。核因子κB （NF-κB）是核转录调节蛋白的一种，具有多向调节的特点；参与多种炎性介质基因和及多种免疫相关基因的转录和调控，是炎症免疫反应中多个信号途径的汇聚点。本课题组前期研究证实NF-κB的核移位是诱导炎症基因表达的关键环节，并在失控性炎症反应（SIRS）和急性肺损伤（ALI）的病理机制中发挥关键作用。但目前拮抗NF-κB活化通路、抑制炎症损伤尚无特效药物和成熟手段。目的：为创伤性ALI筛选精确的基因药物靶点，寻求一种高效、副作用小的炎症靶向控制方法和寻求高效合理的治疗方案提供实验基础和理论依据。方法：本实验运用已成功建立的家兔ALI模型，选定NF-κB信号通路的关键环节为突破口，利用载体/药物技术，对体内外炎症效应细胞（如肺泡巨噬细胞）的NF-κB核移位识别目的基因κB序列的瓶颈‖通路进行特异性靶向封闭；并用分子生物学、免疫学和形态学等方法证实靶向封闭炎症信号通路、调控炎症基因表达防治SIRS/ALI的可行性和有效性。结果：体外实验：脂质体转染的双链寡聚脱氧核苷酸（dsODNs）能有效降低肺泡巨噬细胞中炎症介质IL-1α、IL-6、IL-10、TNF-α和VCAM的mRNA表达和肺泡巨噬细胞培养液中LDH的含量（P0.01）；dsODNs-decoy/脂质体为1:5时转染效率最高，转染6小时后细胞摄取率最高。为体内试验的顺利进行奠定了基础。体内试验：双链寡聚脱氧核苷酸（dsODNs-decoy）能有效减少肺组织的炎症介质IL-1α、IL-6、IL-10、TNF和VCAM的mRNA表达，dsODNs-decoy组与对照组和错配dsODNs-decoy组比较有统计学意义；血清LDH检测实验组明显减少（p0.01）；HE染色显示对照组和错配dsODNs-decoy组中肺泡变形、融合，大小不一；扫描电镜显示对照组和错配dsODNs-decoy组肺泡隔增粗、增厚，肺泡隔不完整、断裂。Western blot显示dsODNs-decoy组中NF-κB p65核移位明显减少。结论：体外实验中脂质体转染dsODNs-decoy具有较高的转染效率，脂质体转染的dsODNs-decoy能有效降低对肺泡巨噬细胞的毒性。体内试验雾化吸入脂质体转染的dsODNs-decoy能有效减轻ALI的症状和炎性介质的表达，并且血清LDH明显降低，预示全身性副反应小。
[Abstract]:Background: most of the studies on ALI/ARDS at home and abroad focus on the imbalance of cytokine network. Although a large number of clinical prevention and treatment of cytokines have been carried out with various drugs, none of them is completely effective and repeatable. Ali is a disorder caused by inflammatory immune signal transduction disorder. When the body is injured by trauma, shock, the lung is the first target organ and one of the source of bacteria. Alveolar macrophages are the activation point of inflammatory waterfall effect in lung tissue. Nuclear factor 魏 B (NF- 魏 B) is a kind of nuclear transcriptional regulatory protein, which is involved in the transcription and regulation of many inflammatory mediators and immune-related genes, and is the convergence point of many signaling pathways in inflammatory immune response. Our previous study confirmed that nuclear translocation of NF- 魏 B is a key link in inducing inflammatory gene expression and plays a key role in the pathological mechanism of uncontrolled inflammatory response (SIRS) and acute lung injury (ALI). However, there are no effective drugs and mature methods to antagonize NF- 魏 B activation pathway and inhibit inflammatory injury. Objective: to provide experimental and theoretical basis for screening accurate gene drug targets for traumatic ALI, seeking a highly effective and less side effect method for the control of inflammatory targets and seeking effective and reasonable therapeutic schemes. Methods: in this experiment, we selected the key link of NF- 魏 B signaling pathway as the breakthrough point and used carrier / drug technology to establish rabbit ALI model successfully. The NF- 魏 B nuclear translocation of inflammatory effector cells (such as alveolar macrophages) in vivo and in vitro was specifically targeted to block the bottleneck pathway of the target gene 魏 B sequence, and was used in molecular biology. Immunological and morphological methods confirmed the feasibility and effectiveness of blocking the inflammatory signaling pathway and regulating the expression of inflammatory genes in the prevention and treatment of SIRS/ALI. Results: in vitro experiment, the double-stranded oligodeoxynucleotide (dsODNs) transfected by liposome could effectively reduce the expression of mRNA and VCAM in the inflammatory mediators IL-1 伪, IL-10, TNF- 伪 and VCAM in alveolar macrophages, and the content of LDH in the culture medium of alveolar macrophages. The transfection efficiency was highest at 1:5. After transfection for 6 hours, the cell uptake rate was the highest. It lays a foundation for the in vivo test. In vivo test: dsODNs-decoy) could effectively reduce the expression of IL-1 伪, IL-6, IL-10, TNF and VCAM mRNA in lung tissue. Compared with the control group and mismatched dsODNs-decoy group, the expression of dsODNs-decoy was significantly lower in the DS-ODNs-decoy group than in the control and mismatched dsODNs-decoy groups. Serum LDH detection significantly reduced alveolar deformation, fusion and size in the control group and mismatched dsODNs-decoy group by HE staining, scanning electron microscopy showed that the alveolar septum was thickened and thickened, and the alveolar septum was incomplete in the control group and mismatched dsODNs-decoy group. Western blot showed that the nuclear translocation of NF- 魏 B p65 was significantly decreased in dsODNs-decoy group. Conclusion: liposome transfection of dsODNs-decoy has a high transfection efficiency in vitro. Liposome transfected dsODNs-decoy can effectively reduce the toxicity to alveolar macrophages. In vivo atomization inhalation of liposome-transfected dsODNs-decoy could effectively alleviate the symptoms of ALI and the expression of inflammatory mediators, and the serum LDH level was significantly decreased, indicating that the systemic side effects were small.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563.8

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