线粒体微粒在创伤性脑损伤相关凝血功能障碍中的作用机制研究

发布时间：2018-05-22 08:00

本文选题：创伤性脑损伤 + 线粒体　；参考：《天津医科大学》2016年博士论文

【摘要】：目的:创伤性脑损伤(traumatic brain injury,TBI)相关凝血功能障碍(traumatic brain injury-associated coagulopathy,TBI-AC)发生和死亡率高,其发生机制尚不明确。课题组前期已发表的研究显示,小鼠TBI后损伤的神经元和神经胶质细胞可释放脑源性微粒(brain derived microparticles,BDMP)通过破坏的血脑屏障进入外周循环血中引起系统性凝血功能障碍(Blood 2015)。本研究将在此基础上,继续研究BDMP的亚型线粒体微粒(mitochondrial microparticles,mt MPs)在TBI后小鼠外周血中的含量及其在TBI-AC中的作用,并通过体外提取mt MPs,检测其对凝血相关细胞的作用。同时检测微粒清除蛋白Lactadherin对mt MPs介导的TBI-AC的治疗作用。本研究将丰富TBI-AC发病机制的理论,并探索新的TBI-AC及TBI继发性损伤的治疗方法。方法:(1)通过小鼠TBI模型,检测小鼠创伤性脑损伤后外周血抗脑抗体的变化。(2)体外实验检测心磷脂(cardiolipin,CL)的促凝活性,并通过尾静脉给予正常小鼠体内注射不同剂量的CL,检测CL在正常小鼠体内的促凝作用。通过扫描电子显微镜(scanning electron microscopy,SEM)观测CL形成胶团结构。(3)通过流式细胞术、RT-PCR和扫描电子显微镜(transmission electron microscopy,TEM)检测小鼠TBI后外周血中mt MPs的释放,同时检测CL与mt MPs的关系。(4)通过流式细胞术,免疫磁珠分选和多步离心法三种不同方法体外提取mt MPs,并通过流式细胞术和TEM对其表型和形态进行鉴定。(5)体外检测mt MPs的促凝作用,并通过小鼠尾静脉给予正常小鼠体内注射mt MPs,通过凝血因子X激活凝血时间实验,ELISA法检测外周血D-dimer含量和组织病理染色,检测mt MPs在正常小鼠体内的促凝作用。(6)通过流式细胞术和RT-PCR检测体外培养的神经元和神经胶质细胞mt MPs的释放。(7)检测mt MPs或CL是否可以激活血小板及其潜在机制。(8)检测mt MPs或CL对血管内皮细胞间屏障完整性和对内皮细胞的激活。(9)体外检测Lactadherin蛋白对mt MPs介导的促凝作用的抑制作用。结果:(1)小鼠TBI后外周血中,在伤后10天开始可检测到抗CL抗体,同时CL胶团在体内及体外具有促凝活性,其在体内可促进小鼠肺组织血管纤维素沉积和微血栓形成。(2)小鼠TBI后6h,检测到脑组织中损伤的神经元和胶质细胞系中线粒体以微粒形式,即线粒体微粒(mt MPs)释放至外周血中,其含量可达到17,547±2,677/μl,mt MPs表面暴露CL。这种mt MPs占到了整个Annexin V+微粒的55.2±12.6%。同时,体外培养的神经细胞系和胶质细胞系在凋亡状态下可释放mt MPs。体外提取的mt MPs可以协同血小板促进内皮细胞的渗漏。(3)正常小鼠经鼠尾静脉注射入mt MPs 30min后,可导致小鼠外周血呈现高凝状态,凝血酶升高,同时可在小鼠肺组织中检测到血管纤维素沉积及肺组织间水肿。(4)mt MPs介导的促凝作用主要是通过线粒体内膜上CL转移至外膜而介导的,其促凝作用可以被微粒清除系统蛋白Lactadherin所阻断,而这种mt MPs上的CL的促凝作用是相同浓度纯化的CL胶团的1600倍。Lactadherin还可以减少经mt MPs注射小鼠的死亡率。结论:(1)TBI后损伤的神经细胞释放mt MPs至外周循环血中,mt MPs表面含有大量CL(2)表面暴露CL的mt MPs具有高的促凝活性,其可以引起TBI后凝血功能障碍的发生。(3)微粒清除蛋白Lactadherin可以抑制CL和mt MPs的促凝作用,其还可以减少注射了mt MPs小鼠的死亡率。本研究揭示了一种新的表面暴露CL的mt MPs介导的TBI相关凝血功能障碍发生的机制,并初步探索了一种潜在的抑制这种促凝机制治疗TBI-AC的方法。
[Abstract]:Objective: the occurrence and mortality of traumatic brain injury (TBI) related coagulation dysfunction (traumatic brain injury-associated coagulopathy, TBI-AC) and high mortality are not clear. Rain derived microparticles, BDMP) causes systemic coagulation dysfunction (Blood 2015) in peripheral circulation by destroying the blood brain barrier (Blood). On this basis, this study will continue to study the content of BDMP subtype mitochondrial particles (mitochondrial microparticles, MT MPs) in the peripheral blood of TBI mice and their role in TBI-AC. The effect of MT MPs on coagulation related cells was detected in vitro. The therapeutic effect of particle scavenging protein Lactadherin on TBI-AC mediated by MT MPs was also detected. This study will enrich the theory of TBI-AC pathogenesis and explore new therapeutic methods for secondary injury of TBI-AC and TBI. Methods: (1) detect mice through mouse TBI model. Change of anti brain antibody in peripheral blood after traumatic brain injury. (2) the activity of cardiolipin (cardiolipin, CL) was detected in vitro, and the normal mice were injected with different doses of CL through the tail vein to detect the procoagulant effect of CL in normal mice. The CL shape was observed by scanning electron microscopy (scanning electron microscopy, SEM). (3) the release of MT MPs in the peripheral blood of mice was detected by flow cytometry, RT-PCR and scanning electron microscopy (transmission electron microscopy, TEM), and the relationship between CL and MT MPs was detected. (4) three different methods were extracted by flow cytometry, immunomagnetic beads and multistep centrifugation. Phenotype and morphology were identified by cytometer and TEM. (5) the procoagulant effect of MT MPs was detected in vitro, MT MPs was injected into normal mice in the tail vein of mice, the coagulation time experiment was activated by coagulation factor X, D-dimer content in peripheral blood and histopathological staining were detected by ELISA method, and the procoagulant activity of MT MPs in normal mice was detected. Use (6) to detect the release of MT MPs in cultured neurons and glial cells by flow cytometry and RT-PCR. (7) whether MT MPs or CL can activate platelets and their potential mechanisms. (8) detection of the intercellular barrier integrity and activation of endothelial cells by MT MPs or CL. (9) detection of Lactadherin protein to MT MPs in vitro Results: (1) the anti CL antibody can be detected in the peripheral blood of mice after TBI, and the anti CL antibody can be detected at the beginning of the injury, and the CL micelles have the procoagulant activity in the body and in vitro, and it can promote the fibrin deposition and the formation of microthrombus in the lungs of the mice in the body. (2) the mice after TBI 6h, detect the injured nerves in the brain tissue. The mitochondria in the cell line and glial cell line are released to the peripheral blood in the form of microparticles, that is, mitochondrial particles (MT MPs). The content of the mitochondria is 17547 + 2677/ Mu L, and the MT MPs on the MT MPs surface is exposed to the MT MPs of the whole Annexin V+ particle, which is 55.2 + 12.6%.. The cultured neural cell lines and glial cell lines can be released in the apoptotic state. MT MPs extracted by S. in vitro can synergistically promote endothelial cell leakage. (3) normal mice injected into the tail vein of MT MPs 30min can lead to hypercoagulable state of peripheral blood and increase of thrombin in mice, and can detect vascular cellulose sedimentation and pulmonary edema in the lung tissue of mice. (4) MT MPs mediated coagulation effect It is mediated mainly through the transfer of the mitochondrial CL to the outer membrane, and its procoagulant action can be blocked by the particle scavenging system protein Lactadherin, and the CL on the MT MPs is 1600 times the CL micelle of the same concentration and can also reduce the mortality of the MT MPs injection mice. Conclusion: (1) after TBI, the injury was damaged. The release of MT MPs on the MT MPs surface containing a large number of CL (2) exposed CL MT MPs has a high procoagulant activity, which can cause the occurrence of coagulating dysfunction after TBI. (3) microparticle scavenging protein Lactadherin can inhibit the coagulation of CL and MT, which can also reduce the mortality of injected mice. The study revealed a new mechanism of MT MPs mediated TBI related coagulation dysfunction mediated by CL, and initially explored a potential method of inhibiting this anticoagulant mechanism for the treatment of TBI-AC.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R651.15

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