外源性一氧化碳釋放分子（CORM-2）對脂多糖刺激后血小板α顆粒釋放的抑制作用及分子機制
發(fā)布時間：2018-09-11 08:59
【摘要】：背景:膿毒癥(Sepsis)是指機體由于感染的失控反應(yīng)所導(dǎo)致的全身性炎癥反應(yīng)綜合征(Systemic Inflammatory Response Syndrome,SIRS),進(jìn)一步可發(fā)展為威脅生命的多臟器功能不全綜合征(Multiple Organ Dysfunction Syndrome,MODS),已成為臨床危重癥主要死亡原因之一。膿毒癥時會發(fā)生凝血系統(tǒng)活化,并促進(jìn)血栓的形成與發(fā)展。血小板α顆粒的釋放在膿毒癥凝血紊亂中發(fā)揮重要作用。我們前期研究證實一氧化碳釋放分子-2(CO-releasing Molecules-2,CORM-2)能顯著抑制LPS刺激下血小板的黏附、聚集、釋放功能。但未見關(guān)于CORM-2對膿毒癥血小板α顆粒釋放的影響的相關(guān)研究。方法:采集健康人靜脈血,建立血小板LPS刺激模型,并使用CORM-2進(jìn)行干預(yù)。酶聯(lián)免疫吸附法(Enzyme Linked Immunosorbent Assay,ELISA)檢測血小板α顆粒內(nèi)容物血小板衍化生長因子(Platelet Derived Growth Factor,PDGF)和基質(zhì)金屬蛋白酶(Matrix Metalloproteinases,MMP)等。流式細(xì)胞術(shù)檢測P-選擇素和整合素αIIbβ3的表達(dá)。免疫熒光顯微鏡(Immunofluorescence Microscope)和透射電子顯微鏡(Transmission Electron Microscope,TEM)下觀察血小板α顆粒的分布。免疫蛋白印跡(Western Blotting,WB)檢測血小板的關(guān)鍵信號分子蛋白激酶Cθ(Protein Kinase Cθ,PKCθ)、Munc18a及其磷酸化的表達(dá)。免疫沉淀反應(yīng)檢測血小板中Munc18a與可溶性N-乙基馬來酰亞胺敏感因子附著蛋白受體(Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor,SNARE)蛋白之間的相互作用。結(jié)果:研究顯示LPS刺激后血小板α顆粒與血小板胞漿膜的融合增加,并促進(jìn)α顆粒內(nèi)容物的釋放;CORM-2能夠有效抑制血小板α顆粒與胞漿膜的融合和α顆粒內(nèi)容物的釋放。進(jìn)一步研究發(fā)現(xiàn)LPS刺激后血小板整合素αIIbβ3的表達(dá)增加,給予CORM-2干預(yù)后,整合素αIIbβ3的表達(dá)減低。同時LPS刺激后血小板PKCθ和Munc18a的磷酸化水平顯著增加,以及SNAREs復(fù)合體形成增加,血小板α顆粒釋放增加。CORM-2干預(yù)后血小板PKCθ和Munc18a的磷酸化水平以及SNAREs復(fù)合體的形成均被抑制。結(jié)論:LPS刺激后血小板α顆粒的釋放增加;CORM-2能夠有效抑制血小板α顆粒的釋放,其機制可能涉及整合素αIIbβ3介導(dǎo)的PKCθ/Munc18a信號途徑的激活。
[Abstract]:Background: sepsis (Sepsis) is a systemic inflammatory response syndrome (Systemic Inflammatory Response Syndrome,SIRS) caused by a runaway response to infection, which can further develop into a life-threatening multiple organ dysfunction syndrome (Multiple Organ Dysfunction Syndrome,MODS), which has become a clinical risk. One of the main causes of severe death. The coagulation system is activated during sepsis and promotes the formation and development of thrombus. Platelet 偽-granule release plays an important role in septic coagulation disorder. Our previous studies have demonstrated that carbon monoxide-2 (CO-releasing Molecules-2,CORM-2) can significantly inhibit platelet adhesion, aggregation and release function induced by LPS. However, there is no related study on the effect of CORM-2 on platelet 偽-granule release in sepsis. Methods: the platelet LPS stimulation model was established by collecting venous blood from healthy people and CORM-2 was used to intervene. Platelet derived growth factor (Platelet Derived Growth Factor,PDGF) and matrix metalloproteinase (Matrix Metalloproteinases,MMP) were detected by enzyme linked immunosorbent assay (Enzyme Linked Immunosorbent Assay,ELISA). The expression of P- selectin and integrin 偽 IIb 尾 3 was detected by flow cytometry. The distribution of platelet 偽-particles was observed under immunofluorescence microscope (Immunofluorescence Microscope) and transmission electron microscope (Transmission Electron Microscope,TEM). The expression of Munc18a and its phosphorylation was detected by Western blot (Western Blotting,WB). The interaction between Munc18a and soluble N-ethylmaleimide receptor (Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor,SNARE) protein in platelets was detected by immunoprecipitation. Results: the results showed that the fusion of platelet 偽 particles with platelet cytosolic membrane was increased after LPS stimulation, and the release of 偽 granule contents was enhanced. Corm 2 could effectively inhibit the fusion of platelet 偽 particles with cytoplasmic membrane and the release of 偽 granule contents. Further study showed that the expression of integrin 偽 IIb 尾 3 increased after LPS stimulation, and decreased after CORM-2 intervention. At the same time, the phosphorylation levels of PKC 胃 and Munc18a, the formation of SNAREs complex, the release of 偽 -platelet particles, the phosphorylation of PKC 胃 and Munc18a and the formation of SNAREs complex were inhibited after LPS stimulation. Conclusion the increased release of platelet 偽 particles induced by FPS can effectively inhibit the release of platelet 偽 particles. The mechanism may be related to the activation of integrin 偽 IIb 尾 3 mediated PKC 胃 / Munc18a signaling pathway.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R459.7
本文編號：2236230
[Abstract]:Background: sepsis (Sepsis) is a systemic inflammatory response syndrome (Systemic Inflammatory Response Syndrome,SIRS) caused by a runaway response to infection, which can further develop into a life-threatening multiple organ dysfunction syndrome (Multiple Organ Dysfunction Syndrome,MODS), which has become a clinical risk. One of the main causes of severe death. The coagulation system is activated during sepsis and promotes the formation and development of thrombus. Platelet 偽-granule release plays an important role in septic coagulation disorder. Our previous studies have demonstrated that carbon monoxide-2 (CO-releasing Molecules-2,CORM-2) can significantly inhibit platelet adhesion, aggregation and release function induced by LPS. However, there is no related study on the effect of CORM-2 on platelet 偽-granule release in sepsis. Methods: the platelet LPS stimulation model was established by collecting venous blood from healthy people and CORM-2 was used to intervene. Platelet derived growth factor (Platelet Derived Growth Factor,PDGF) and matrix metalloproteinase (Matrix Metalloproteinases,MMP) were detected by enzyme linked immunosorbent assay (Enzyme Linked Immunosorbent Assay,ELISA). The expression of P- selectin and integrin 偽 IIb 尾 3 was detected by flow cytometry. The distribution of platelet 偽-particles was observed under immunofluorescence microscope (Immunofluorescence Microscope) and transmission electron microscope (Transmission Electron Microscope,TEM). The expression of Munc18a and its phosphorylation was detected by Western blot (Western Blotting,WB). The interaction between Munc18a and soluble N-ethylmaleimide receptor (Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor,SNARE) protein in platelets was detected by immunoprecipitation. Results: the results showed that the fusion of platelet 偽 particles with platelet cytosolic membrane was increased after LPS stimulation, and the release of 偽 granule contents was enhanced. Corm 2 could effectively inhibit the fusion of platelet 偽 particles with cytoplasmic membrane and the release of 偽 granule contents. Further study showed that the expression of integrin 偽 IIb 尾 3 increased after LPS stimulation, and decreased after CORM-2 intervention. At the same time, the phosphorylation levels of PKC 胃 and Munc18a, the formation of SNAREs complex, the release of 偽 -platelet particles, the phosphorylation of PKC 胃 and Munc18a and the formation of SNAREs complex were inhibited after LPS stimulation. Conclusion the increased release of platelet 偽 particles induced by FPS can effectively inhibit the release of platelet 偽 particles. The mechanism may be related to the activation of integrin 偽 IIb 尾 3 mediated PKC 胃 / Munc18a signaling pathway.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R459.7
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1 莊明峰;外源性一氧化碳釋放分子（CORM-2）對脂多糖刺激后血小板α顆粒釋放的抑制作用及分子機制[D];江蘇大學(xué);2016年
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