血浆高分子量激肽原在内毒素血症中致病作用的初步研究
发布时间：2018-08-26 12:50
【摘要】：【研究背景和意义】血浆高分子量激肽原（high molecular weight kininogen,HK）是血浆激肽释放酶-激肽系统（Plasma kallikrein-kinin system, KKS）的主要成分之一。临床研究表明，败血症患者中KKS被活化，而且HK被裂解，说明HK参与败血症的发病过程。但是，迄今对于HK在内毒素血症发病中的作用完全不清楚。本研究使用遗传学方法，对HK在内毒素血症中的作用进行了系列研究。 【研究目的】认识HK在内毒素血症中的致病作用及其对天然免疫的调控机制。 【研究方法】Kng1~(-/-)小鼠和其同窝对照野生型小鼠腹腔内注射内毒素(lipopolysaccharide，LPS)，观察小鼠的生存率；应用ELISA检测血浆中缓激肽（BK）和炎症因子的含量；应用实时PCR检测白细胞和单核细胞炎症因子mRNA水平；制作主要器官的组织切片和扫描电镜，观察组织的病理改变；检测支气管肺泡灌洗液中炎性指标变化。 【研究结果】(1)应用Western和RT-PCR发现Kng1~(-/-)小鼠血浆中HK蛋白和肝脏中HK mRNA表达均缺陷。Kng1~(-/-)小鼠血浆活化部分凝血激酶时间与野生型小鼠相比显著延长(56.81±5.428v.s.33.64±2.288seconds, p=0.0004)。(2) Kng1~(-/-)小鼠和野生型小鼠腹腔内注射LPS(50mg/kg)48小时后，Kng1~(-/-)小鼠的存活率比野生型小鼠明显提高(100%v.s.10%, p0.001)，提示Kng1基因的缺陷对LPS导致的死亡具有保护作用。(3)野生型小鼠在注射LPS3小时后，小鼠血浆中缓激肽含量比0小时明显增高(7896±41.65v.s.4476±15.14pg/mL, p=0.0026)，提示LPS在体内可以导致KKS系统的活化，裂解HK产生缓激肽。(4)在LPS注射4小时后，Kng1~(-/-)小鼠血浆中炎症因子的水平比野生型小鼠显著降低(TNF-α:213.4±100.7v.s.760.3±185.8pg/mL，p=0.0322；IL-1β:138.9±27.19v.s519.3±93.9pg/mL, p=0.0092；IL-6:429.4±51.11v.s722.1±96.49pg/mL, p=0.0231)。(5)使用流式细胞仪分选出CD90, B220, CD49b, NK1.1, Ly-6G阴性表达和CD11b阳性表达的单核细胞，实时PCR检测单核细胞炎症因子mRNA水平。与野生型小鼠相比，Kng1~(-/-)小鼠单核细胞炎症因子mRNA水平显著降低(IL-1β:0.622±0.077v.s.2.674±0.174, p=0.0095;IL-6:0.454±0.048v.s.3.255±1.028,p=0.0004)。(6)通过HE染色，电镜观察和检测支气管肺泡灌洗液评价小鼠急性肺损伤程度。从HE染色和电镜中可以看出，野生型小鼠的肺组织间质增生明显，纤维细胞增多，炎性细胞浸润明显，血管扩张，提示出现典型的肺组织急性损伤，而Kng1~(-/-)小鼠上述肺组织病理表现明显改善。与野生型小鼠相比，，Kng1~(-/-)小鼠支气管肺泡灌洗液中蛋白含量(0.0436±0.01341v.s.0.1062±0.0093ug/ul, p=0.0188)和总细胞数(0.7667×10~4±881v.s.1.6×10~4±2640个,p=0.0404)明显降低，肺组织髓过氧化物酶活性也明显降低(680.5±116.2v.s.1791±327.3ng/mL, p=0.0127)。上述观察提示Kng1基因的缺陷缓解内毒素导致的急性肺损伤。(7)通过HE染色观察小鼠的肝和肾发现，野生型小鼠的肝和肾都出现不同程度的损伤，而Kng1~(-/-)小鼠的肝和肾组织损伤明显改善，说明Kng1基因的缺陷可以缓解内毒素导致的肝肾组织损伤。 【结论】本研究说明HK是导致内毒素血症发病的一种关键炎症介质。
[Abstract]:[BACKGROUND AND SIGNIFICANCE] Plasma high molecular weight kininogen (HK) is one of the main components of plasma kallikrein-kinin system (KKS). So far, the role of HK in the pathogenesis of endotoxemia is completely unclear. This study used genetic methods to study the role of HK in endotoxemia.
[Objective] to understand the pathogenic role of HK in endotoxemia and its regulation mechanism on innate immunity.
[Methods] Kng1 ~(-/-) mice and wild type mice in the same litter were injected with lipopolysaccharide (LPS) intraperitoneally to observe the survival rate of mice, the levels of bradykinin (BK) and inflammatory factors in plasma were detected by ELISA, the levels of inflammatory factors mRNA in leukocytes and monocytes were detected by real-time PCR, and the main organs were made. Tissue sections and scanning electron microscopy were used to observe the pathological changes of the tissues and detect the changes of inflammatory indexes in bronchoalveolar lavage fluid.
[Results] The expression of HK protein in plasma and HK mRNA in liver of Kng1 ~(-/-) mice were found to be deficient by Western and RT-PCR. The activated partial thrombokinase time in plasma of Kng1 ~(-/-) mice was significantly longer than that of wild type mice (56.81 (5.428) v.s.33.64 (2.288) seconds, P = 0.0004). (2) Kng1 (-/-) mice and wild type mice. The survival rate of Kng1 ~(-/-) mice was significantly higher than that of wild-type mice 48 hours after LPS injection (100% v.s.10%, P 0.001), suggesting that the deficiency of Kng1 gene has a protective effect on LPS-induced death. (3) Bradykinin content in plasma of wild-type mice was significantly higher than that of 0 hours after LPS injection (7896 65v.s.4476 (15.14p) G / mL, P = 0.0026), suggesting that LPS can activate the KKS system in vivo and produce bradykinin by lysis of HK. (4) After 4 hours of LPS injection, the levels of inflammatory factors in plasma of Kng1 ~(-/-) mice were significantly lower than those of wild type mice (TNF-a: 213.4 ~ (-/-) 100.7 v. s. 760.3 ~ 185.8 p g / mL, P = 0.0322; IL-1 beta: 138.9 ~ (- 27.19 v. s519.3 (- 93.9 p g / mL), P = 0.0092; (5) Monocytes with negative expression of CD90, B220, CD49b, NK1.1, Ly-6G and positive expression of CD11b were separated by flow cytometry, and the levels of inflammatory cytokine mRNA in monocytes were detected by real-time PCR. (6) The degree of acute lung injury in mice was evaluated by HE staining, electron microscope observation and detection of bronchoalveolar lavage fluid. Compared with the wild type mice, the protein content in bronchoalveolar lavage fluid (0.0436 (-/-) mice was (0.01341 v.s. ~4卤2640涓
本文编号：2204921
[Abstract]:[BACKGROUND AND SIGNIFICANCE] Plasma high molecular weight kininogen (HK) is one of the main components of plasma kallikrein-kinin system (KKS). So far, the role of HK in the pathogenesis of endotoxemia is completely unclear. This study used genetic methods to study the role of HK in endotoxemia.
[Objective] to understand the pathogenic role of HK in endotoxemia and its regulation mechanism on innate immunity.
[Methods] Kng1 ~(-/-) mice and wild type mice in the same litter were injected with lipopolysaccharide (LPS) intraperitoneally to observe the survival rate of mice, the levels of bradykinin (BK) and inflammatory factors in plasma were detected by ELISA, the levels of inflammatory factors mRNA in leukocytes and monocytes were detected by real-time PCR, and the main organs were made. Tissue sections and scanning electron microscopy were used to observe the pathological changes of the tissues and detect the changes of inflammatory indexes in bronchoalveolar lavage fluid.
[Results] The expression of HK protein in plasma and HK mRNA in liver of Kng1 ~(-/-) mice were found to be deficient by Western and RT-PCR. The activated partial thrombokinase time in plasma of Kng1 ~(-/-) mice was significantly longer than that of wild type mice (56.81 (5.428) v.s.33.64 (2.288) seconds, P = 0.0004). (2) Kng1 (-/-) mice and wild type mice. The survival rate of Kng1 ~(-/-) mice was significantly higher than that of wild-type mice 48 hours after LPS injection (100% v.s.10%, P 0.001), suggesting that the deficiency of Kng1 gene has a protective effect on LPS-induced death. (3) Bradykinin content in plasma of wild-type mice was significantly higher than that of 0 hours after LPS injection (7896 65v.s.4476 (15.14p) G / mL, P = 0.0026), suggesting that LPS can activate the KKS system in vivo and produce bradykinin by lysis of HK. (4) After 4 hours of LPS injection, the levels of inflammatory factors in plasma of Kng1 ~(-/-) mice were significantly lower than those of wild type mice (TNF-a: 213.4 ~ (-/-) 100.7 v. s. 760.3 ~ 185.8 p g / mL, P = 0.0322; IL-1 beta: 138.9 ~ (- 27.19 v. s519.3 (- 93.9 p g / mL), P = 0.0092; (5) Monocytes with negative expression of CD90, B220, CD49b, NK1.1, Ly-6G and positive expression of CD11b were separated by flow cytometry, and the levels of inflammatory cytokine mRNA in monocytes were detected by real-time PCR. (6) The degree of acute lung injury in mice was evaluated by HE staining, electron microscope observation and detection of bronchoalveolar lavage fluid. Compared with the wild type mice, the protein content in bronchoalveolar lavage fluid (0.0436 (-/-) mice was (0.01341 v.s. ~4卤2640涓
本文编号：2204921
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