【作者】 王宁；

【导师】 王海嵘；

【作者基本信息】 上海交通大学 ， 急诊医学， 2020， 硕士

【摘要】 目的:血管内皮屏障损伤是脓毒症病理生理过程的中心环节。氯喹(Chloroquine,CQ)具有多种药理作用,近来研究发现,氯喹能够改善脓毒症小鼠的肺损伤,但对内皮细胞屏障损伤的作用尚未见报道。本研究通过体外细胞水平初步探讨氯喹对脂多糖(Lipopolysaccharide,LPS)诱导的内皮细胞屏障损伤的作用及机制。方法:1.通过FITC-葡聚糖渗漏法检测细胞通透性,免疫荧光法检测细胞骨架形态变化,western blot法检测细胞连接相关蛋白的表达,初步明确氯喹对LPS诱导的内皮细胞屏障损伤的作用;2.通过CCK-8法检测细胞活性,Annexin V FITC/PI双染法检测细胞凋亡,用Elisa和PCR法检测细胞因子的分泌及表达,western blot法检测相关蛋白的表达,探讨氯喹的保护机制;3.加或不加PI3K抑制剂LY294002,探究PI3K/Akt信号通路在氯喹对内皮细胞屏障保护中的作用。结果:1.与LPS组相比,氯喹减轻FITC-葡聚糖的渗漏,同时稳定细胞骨架,上调VE-cadherin、ZO-1、occludin的表达,对LPS诱导的内皮细胞屏障损伤具有抑制作用;2.氯喹通过提高内皮细胞活性,降低内皮细胞凋亡率并升高Bcl-2/Bax比值、下调活化的caspase3蛋白水平,抑制炎症因子产生及NF-κB活化,上调p-Akt/Akt蛋白的表达,减轻内皮细胞屏障损伤;3.LY294002逆转氯喹对内皮细胞屏障的保护作用。结论:氯喹通过激活PI3K/Akt信号通路减轻LPS诱导的内皮细胞屏障损伤。更多还原

【Abstract】 Objective: Vascular endothelial barrier dysfunction is a central event in the pathogenesis of sepsis.Chloroquine(CQ)has been shown to have various effects,and currently has established protective roles in acute lung injury of rat induced by sepsis,however,its effects on endothelial barrier dysfunction have not been reported.This study aimed to investigate the effects of chloroquine on the endothelial barrier injury induced by lipopolysaccharide(LPS)in vitro.Methods: 1.The endothelial permeability was detected by FITC dextran leakage,cytoskeletons were subjected to immunofluorescence staining,expressions of cell junction related proteins were analyzed by western blot,thus the effect of chloroquine on LPS-induced endothelial cell barrier injury was preliminarily determined;2.To explore the mechanism,cell viability was evaluated by CCK-8,cell apoptosis was evaluated by Annexin V FITC/PI staining,the extracellular levels of TNF-α and IL-6were determined by enzyme-linked immunosorbent assay(Elisa),the m RNA expression of TNF-α,IL-6,ICAM-1,VCAM-1 was measured by real-time quantitative polymerase chain reaction(q PCR),protein expression were analyzed by western blot;3.To explore the role of PI3 K / Akt signal pathway in the protective mechanism of endothelial barrier by chloroquine.Results: 1.Compared with LPS group,chloroquine reduced the leakage of FITCdextran,stabilized the cytoskeleton,upregulated the expression of VE-cadherin,ZO-1and occludin,which indicates its protective role in LPS-induced endothelial barrier damage;2.Chloroquine showed benefit effects on endothelial cell barrier damage: the apoptosis rate and the level of activated caspase 3 protein were decreased,the ratio of Bcl-2 / Bax was increased,the production of inflammatory factors and NF-κ B activation were also inhibited,besides,p-Akt / Akt protein ratio was up-regulated;3.The above protective effects of chloroquine on endothelial cells were reversed by LY294002.Conclusions: CQ attenuated LPS-induced endothelial barrier injury partially via PI3K/Akt signaling pathway.更多还原