线粒体DNA激活内皮细胞引起急性肺损伤的实验研究

发布时间：2018-05-17 04:37

本文选题：mtDNA + 肺微血管内皮细胞　；参考：《中国人民解放军医学院》2013年博士论文

【摘要】：目的研究线粒体DNA(mitochondrial DNA, mtDNA)对大鼠肺微血管内皮细胞活化和通透性的影响及其分子生物学机制,探索创伤后组织细胞破裂释放的线粒体DNA导致系统性炎症及急性肺损伤的机制,为创伤后SIRS及急性肺损伤的救治提供实验依据。方法本研究采用分子生物学、流式细胞仪等技术,探索mtDNA作为一利,源头性损伤相关分子模式直接激活内皮细胞,参与启动全身炎症反应综合征(systemic inflammatory response syndrome,SIRS)的分子机制,探索线粒体DNA启动、转导、调控失控性炎症反应机制。通过大鼠尾静脉注入临床浓度的mtDNA导致急性肺损伤,检测外周血炎症因子TNF-α、IL-6及血管性血友病因子(von Willebrand factor, vWF)、sE-selectin和sICAM-1浓度,检测肺血管通透性；以临床浓度mtDNA刺激体外培养大鼠肺微血管内皮细胞,检测其通透性变化及vWF、sE-selectin和sICAM-1表达水平以及TLR-9mRNA、NF-κB亚单位p65mRNA、I-κB mRNA表达水平；将大鼠肺微血管内皮细胞与中性粒细胞共培养,予mtDNA刺激后观察微血管内皮细胞存活率、中性粒细胞粘附情况。结果10ug/ml mtDNA体内注射导致大鼠急性肺损伤；mtDNA刺激大鼠微血管内皮细胞中的TLR-9mRNA、NF-κB亚单位p65mRNA表达增加,I-κ BmRNA表达降低：mtDNA能直接激活大鼠微血管内皮细胞,显著增加vwF、sE-selectin和sICAM-1表达,使大鼠微血管内皮细胞通透性增加,显著促进大鼠微血管内皮细胞与中性粒细胞的粘附。结论mtDNA能够通过TLR-9激活大鼠微血管内皮细胞NF-κB通路,刺激大鼠微血管内皮细胞产生vwF、sE-selectin和sICAM-1,增加内皮细胞通透性,显著促进与中性粒细胞的粘附。mtDNA激活内皮细胞是创伤后SIRS及ALI的重要启动机制。
[Abstract]:Objective to investigate the effect of mitochondrial DNA(mitochondrial DNA (mtDNA) on the activation and permeability of rat pulmonary microvascular endothelial cells and its molecular biological mechanism, and to explore the mechanism of systemic inflammation and acute lung injury induced by mitochondrial DNA released by tissue cell rupture after trauma. To provide experimental evidence for the treatment of SIRS and acute lung injury after trauma. Methods in this study, molecular biology, flow cytometry and other techniques were used to explore the molecular mechanism of mtDNA, as a beneficial molecular model associated with source injury, directly activating endothelial cells and initiating systemic inflammatory response syndromes (SIRs). To explore the mechanism of mitochondrial DNA activation, transduction and control of uncontrolled inflammatory response. Acute lung injury was induced by injecting clinical concentration of mtDNA into the tail vein of rats. The levels of TNF- 伪 IL-6, von Willebrand factor, vWFU E-selectin and sICAM-1 in peripheral blood were measured to determine the pulmonary vascular permeability. Pulmonary microvascular endothelial cells were cultured in vitro with clinical concentration of mtDNA. The permeability, the expression of vWFN E-selectin and sICAM-1 and the expression of TLR-9mRNA-NF- 魏 B mRNA were detected, and the p65mRNA-kappa B subunit p65mRNA- 魏 B mRNA were co-cultured with neutrophils. The survival rate of microvascular endothelial cells and the adhesion of neutrophils were observed after mtDNA stimulation. Results 10ug/ml mtDNA injection in vivo stimulated the expression of TLR-9 mRNA-NF- 魏 B subunit p65mRNA in rat microvascular endothelial cells (MECs) and increased the expression of TLR-9mRNA-kappa B subunit p65mRNA. The expression of TLR-9mRNA-kappa B subunit in rat microvascular endothelial cells (MECs) and the expression of VWFNs E-selectin and sICAM-1 were directly activated by 10ug/ml mtDNA. The permeability of rat microvascular endothelial cells was increased, and the adhesion of rat microvascular endothelial cells to neutrophils was significantly promoted. Conclusion mtDNA can activate the NF- 魏 B pathway of rat microvascular endothelial cells through TLR-9, stimulate the production of vwFN E-selectin and sICAM-1 by rat microvascular endothelial cells, and increase the permeability of endothelial cells. Promoting the adhesion with neutrophil. Mt DNA activated endothelial cells is an important mechanism of SIRS and ALI after trauma.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563

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