【摘要】：【研究背景和意義】血漿高分子量激肽原（high molecular weight kininogen,HK）是血漿激肽釋放酶-激肽系統(tǒng)（Plasma kallikrein-kinin system, KKS）的主要成分之一。臨床研究表明，敗血癥患者中KKS被活化，而且HK被裂解，說明HK參與敗血癥的發(fā)病過程。但是，迄今對(duì)于HK在內(nèi)毒素血癥發(fā)病中的作用完全不清楚。本研究使用遺傳學(xué)方法，對(duì)HK在內(nèi)毒素血癥中的作用進(jìn)行了系列研究。 【研究目的】認(rèn)識(shí)HK在內(nèi)毒素血癥中的致病作用及其對(duì)天然免疫的調(diào)控機(jī)制。 【研究方法】Kng1~(-/-)小鼠和其同窩對(duì)照野生型小鼠腹腔內(nèi)注射內(nèi)毒素(lipopolysaccharide，LPS)，觀察小鼠的生存率；應(yīng)用ELISA檢測(cè)血漿中緩激肽（BK）和炎癥因子的含量；應(yīng)用實(shí)時(shí)PCR檢測(cè)白細(xì)胞和單核細(xì)胞炎癥因子mRNA水平；制作主要器官的組織切片和掃描電鏡，觀察組織的病理改變；檢測(cè)支氣管肺泡灌洗液中炎性指標(biāo)變化。 【研究結(jié)果】(1)應(yīng)用Western和RT-PCR發(fā)現(xiàn)Kng1~(-/-)小鼠血漿中HK蛋白和肝臟中HK mRNA表達(dá)均缺陷。Kng1~(-/-)小鼠血漿活化部分凝血激酶時(shí)間與野生型小鼠相比顯著延長(zhǎng)(56.81±5.428v.s.33.64±2.288seconds, p=0.0004)。(2) Kng1~(-/-)小鼠和野生型小鼠腹腔內(nèi)注射LPS(50mg/kg)48小時(shí)后，Kng1~(-/-)小鼠的存活率比野生型小鼠明顯提高(100%v.s.10%, p0.001)，提示Kng1基因的缺陷對(duì)LPS導(dǎo)致的死亡具有保護(hù)作用。(3)野生型小鼠在注射LPS3小時(shí)后，小鼠血漿中緩激肽含量比0小時(shí)明顯增高(7896±41.65v.s.4476±15.14pg/mL, p=0.0026)，提示LPS在體內(nèi)可以導(dǎo)致KKS系統(tǒng)的活化，裂解HK產(chǎn)生緩激肽。(4)在LPS注射4小時(shí)后，Kng1~(-/-)小鼠血漿中炎癥因子的水平比野生型小鼠顯著降低(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)使用流式細(xì)胞儀分選出CD90, B220, CD49b, NK1.1, Ly-6G陰性表達(dá)和CD11b陽性表達(dá)的單核細(xì)胞，實(shí)時(shí)PCR檢測(cè)單核細(xì)胞炎癥因子mRNA水平。與野生型小鼠相比，Kng1~(-/-)小鼠單核細(xì)胞炎癥因子mRNA水平顯著降低(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)通過HE染色，電鏡觀察和檢測(cè)支氣管肺泡灌洗液評(píng)價(jià)小鼠急性肺損傷程度。從HE染色和電鏡中可以看出，野生型小鼠的肺組織間質(zhì)增生明顯，纖維細(xì)胞增多，炎性細(xì)胞浸潤(rùn)明顯，血管擴(kuò)張，提示出現(xiàn)典型的肺組織急性損傷，而Kng1~(-/-)小鼠上述肺組織病理表現(xiàn)明顯改善。與野生型小鼠相比，，Kng1~(-/-)小鼠支氣管肺泡灌洗液中蛋白含量(0.0436±0.01341v.s.0.1062±0.0093ug/ul, p=0.0188)和總細(xì)胞數(shù)(0.7667×10~4±881v.s.1.6×10~4±2640個(gè),p=0.0404)明顯降低，肺組織髓過氧化物酶活性也明顯降低(680.5±116.2v.s.1791±327.3ng/mL, p=0.0127)。上述觀察提示Kng1基因的缺陷緩解內(nèi)毒素導(dǎo)致的急性肺損傷。(7)通過HE染色觀察小鼠的肝和腎發(fā)現(xiàn)，野生型小鼠的肝和腎都出現(xiàn)不同程度的損傷，而Kng1~(-/-)小鼠的肝和腎組織損傷明顯改善，說明Kng1基因的缺陷可以緩解內(nèi)毒素導(dǎo)致的肝腎組織損傷。 【結(jié)論】本研究說明HK是導(dǎo)致內(nèi)毒素血癥發(fā)病的一種關(guān)鍵炎癥介質(zhì)。
[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鹵2640涓

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