外源性一氧化碳释放分子（CORM-2）对脂多糖刺激后血小板α颗粒释放的抑制作用及分子机制
发布时间：2018-09-11 08:59
【摘要】：背景:脓毒症(Sepsis)是指机体由于感染的失控反应所导致的全身性炎症反应综合征(Systemic Inflammatory Response Syndrome,SIRS),进一步可发展为威胁生命的多脏器功能不全综合征(Multiple Organ Dysfunction Syndrome,MODS),已成为临床危重症主要死亡原因之一。脓毒症时会发生凝血系统活化,并促进血栓的形成与发展。血小板α颗粒的释放在脓毒症凝血紊乱中发挥重要作用。我们前期研究证实一氧化碳释放分子-2(CO-releasing Molecules-2,CORM-2)能显著抑制LPS刺激下血小板的黏附、聚集、释放功能。但未见关于CORM-2对脓毒症血小板α颗粒释放的影响的相关研究。方法:采集健康人静脉血,建立血小板LPS刺激模型,并使用CORM-2进行干预。酶联免疫吸附法(Enzyme Linked Immunosorbent Assay,ELISA)检测血小板α颗粒内容物血小板衍化生长因子(Platelet Derived Growth Factor,PDGF)和基质金属蛋白酶(Matrix Metalloproteinases,MMP)等。流式细胞术检测P-选择素和整合素αIIbβ3的表达。免疫荧光显微镜(Immunofluorescence Microscope)和透射电子显微镜(Transmission Electron Microscope,TEM)下观察血小板α颗粒的分布。免疫蛋白印迹(Western Blotting,WB)检测血小板的关键信号分子蛋白激酶Cθ(Protein Kinase Cθ,PKCθ)、Munc18a及其磷酸化的表达。免疫沉淀反应检测血小板中Munc18a与可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor,SNARE)蛋白之间的相互作用。结果:研究显示LPS刺激后血小板α颗粒与血小板胞浆膜的融合增加,并促进α颗粒内容物的释放;CORM-2能够有效抑制血小板α颗粒与胞浆膜的融合和α颗粒内容物的释放。进一步研究发现LPS刺激后血小板整合素αIIbβ3的表达增加,给予CORM-2干预后,整合素αIIbβ3的表达减低。同时LPS刺激后血小板PKCθ和Munc18a的磷酸化水平显著增加,以及SNAREs复合体形成增加,血小板α颗粒释放增加。CORM-2干预后血小板PKCθ和Munc18a的磷酸化水平以及SNAREs复合体的形成均被抑制。结论:LPS刺激后血小板α颗粒的释放增加;CORM-2能够有效抑制血小板α颗粒的释放,其机制可能涉及整合素αIIbβ3介导的PKCθ/Munc18a信号途径的激活。
[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 伪-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 伪-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 伪 IIb 尾 3 was detected by flow cytometry. The distribution of platelet 伪-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 伪 particles with platelet cytosolic membrane was increased after LPS stimulation, and the release of 伪 granule contents was enhanced. Corm 2 could effectively inhibit the fusion of platelet 伪 particles with cytoplasmic membrane and the release of 伪 granule contents. Further study showed that the expression of integrin 伪 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 伪 -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 伪 particles induced by FPS can effectively inhibit the release of platelet 伪 particles. The mechanism may be related to the activation of integrin 伪 IIb 尾 3 mediated PKC 胃 / Munc18a signaling pathway.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R459.7
本文编号：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 伪-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 伪-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 伪 IIb 尾 3 was detected by flow cytometry. The distribution of platelet 伪-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 伪 particles with platelet cytosolic membrane was increased after LPS stimulation, and the release of 伪 granule contents was enhanced. Corm 2 could effectively inhibit the fusion of platelet 伪 particles with cytoplasmic membrane and the release of 伪 granule contents. Further study showed that the expression of integrin 伪 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 伪 -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 伪 particles induced by FPS can effectively inhibit the release of platelet 伪 particles. The mechanism may be related to the activation of integrin 伪 IIb 尾 3 mediated PKC 胃 / Munc18a signaling pathway.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R459.7
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1 庄明峰;外源性一氧化碳释放分子（CORM-2）对脂多糖刺激后血小板α颗粒释放的抑制作用及分子机制[D];江苏大学;2016年
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