神经细胞来源Microparticles在颅脑创伤相关凝血功能障碍发生中的机制研究

发布时间：2018-09-14 20:48

【摘要】：目的：目前国际上尚缺乏颅脑创伤(traumatic brain injury, TBI)相关凝血功能障碍明确的发生机制及有效的治疗方法。本实验将通过建立中型TBI小鼠模型,观察循环血中神经细胞来源microparticles (brain-derived microparticles, BDMP)含量及凝血功能变化,结合体外制备BDMP的方法,体外研究BDMP的促凝作用,与血小板的相互作用及Lactadherin蛋白对BDMP的清除作用。试图阐明TBI相关凝血功能障碍的物质基础,明确TBI相关凝血功能障碍的发生机制,探讨建立预防治疗TBI引起的凝血功能障碍的新方法,成为研究TBI相关凝血功能障碍机制的新方向。方法：1)建立中型TBI小鼠模型,应用流式细胞仪和活化因子X凝血时间实验,检测TBI小鼠循环血中BDMP含量及凝血功能的变化；2)应用脑组织匀浆分步离心的方法体外制备BDMP;3)应用透射电镜、流式细胞仪和Western Blot方法鉴定所得BDMP的形态学及表型；4)应用活化因子X凝血时间实验和凝血酶生成实验,体外检测BDMP的促凝作用；5)经鼠尾静脉注射BDMP到正常小鼠体内,应用活化因子X凝血时间实验、血浆纤维蛋白原水平测定和病理PTAH染色方法,体内检测BDMP的促凝作用；6)应用流式细胞仪和扫描电镜观察BDMP与血小板的结合情况；7)应用流式细胞仪检测BDMP对血小板的活化作用。8)应用Transwell小室系统检测BDMP在血小板介导下穿透HUVEC细胞的能力；9)应用流式细胞仪和活化因子X凝血时间实验检测Lactadherin蛋白对BDMP的清除作用。结果：1)TBI小鼠循环血中BDMP呈现先增高后降低的趋势,在TBI后3h时其表达达到顶峰。小鼠TBI后3h的贫血小板血浆(platelet poor plasma, PPP)其凝血时间明显缩短,此时PPP中BDMP的表达最高。而在经过超速离心将BDMP去除后,无MP血浆(MP-free plasma, MPFP)的凝血时间明显延长；2)透射电镜下BDMP具有完整的膜结构,且表面表达大量的胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)。流式细胞及Western Blot结果显示BDMP表达大量的神经元和胶质细胞特异性标记,而不表达血小板、红细胞、白细胞和内皮细胞的标志物。BDMP上磷脂酰丝氨酸(PS)和组织因子在体外具有促凝作用。最后将BDMP注射到正常小鼠体内,小鼠PPP的凝血时间明显延长,血浆纤维蛋白原明显降低,组织中纤维素沉积明显；3)流式细胞仪和扫描电镜观察至BDMP能够与血小板结合,其结合后可以促进血小板表面PS的表达增加,同时可以激活血小板,导致血小板活化标志P-选择素表达增加和钙离子内流增加。反过来,激活的血小板可以介导BDMP穿透激活的HUVEC细胞；4)Lactadherin蛋白可以和BDMP结合,并在结合后减弱BDMP的促凝作用。结论：1)TBI小鼠循环血中检测到具有促凝活性的BDMP,可能是发生TBI相关凝血功能障碍的物质基础；2)通过脑组织匀浆分步离心的方法在体外成功制备出了大量的、纯度高的BDMP,为后续的体外实验提供了充足的原料；3)BDMP能够激活血小板,而激活的血小板可以协助BDMP穿透BBB,部分解释了TBI相关凝血功能障碍的发生机制；4)BDMP可与Lactadherin蛋白结合,能够抑制BDMP的促凝活性,该BDMP的清除机制,为TBI合并凝血功能障碍的治疗提供了一条新思路。
[Abstract]:OBJECTIVE: There is no definite mechanism and effective treatment for traumatic brain injury (TBI) related coagulation dysfunction in the world. In this study, we will establish a medium-sized TBI mouse model to observe the content of brain-derived microparticles (BDMP) in circulating blood and the coagulation function. In order to clarify the material basis of TBI-related coagulation dysfunction, to clarify the mechanism of TBI-related coagulation dysfunction, and to explore the prevention and treatment of TBI-induced coagulation dysfunction. The new method has become a new direction for studying the mechanism of TBI related coagulation dysfunction.
Methods: 1) To establish a medium-sized TBI mice model and detect the changes of BDMP content and coagulation function in circulating blood of TBI mice by flow cytometry and activating factor X coagulation time test; 2) To prepare BDMP by step centrifugation of brain tissue homogenate in vitro; 3) To identify the morphology of BDMP by transmission electron microscopy, flow cytometry and Western Blot method. 4) The coagulation-promoting effect of BDMP was detected in vitro by coagulation time test and thrombin production test with activator X. 5) The coagulation-promoting effect of BDMP was detected in vivo by coagulation time test with activator X, plasma fibrinogen level and pathological PTAH staining after injecting BDMP into the tail vein of mice. Flow cytometry and scanning electron microscopy were used to observe the binding of BDMP to platelets; 7) Flow cytometry was used to detect the activation of BDMP on platelets; 8) Transwell chamber system was used to detect the ability of BDMP to penetrate HUVEC cells mediated by platelets; 9) Flow cytometry and activating factor X coagulation time test were used to detect Lactadheri. The scavenging effect of N protein on BDMP.
Results: 1) BDMP expression in circulating blood of TBI mice increased at first and then decreased, and reached its peak at 3 hours after TBI. The platelet poor plasma (PPP) of mice at 3 hours after TBI significantly shortened the coagulation time, and BDMP expression was the highest in PPP. The results of flow cytometry and Western Blot showed that BDMP expressed a lot of specific markers of neurons and glia cells, but not platelets, red blood cells. Leukocyte and endothelial cell markers. Phosphatidylserine (PS) and tissue factor on BDMP can promote coagulation in vitro. At last, BDMP was injected into normal mice. The coagulation time of PPP in mice was prolonged, plasma fibrinogen was decreased, and cellulose deposition was observed by flow cytometry and scanning electron microscopy. In turn, activated platelets can mediate BDMP penetration through activated HUVEC cells; 4) Lactadherin can bind to BDMP. The procoagulant action of BDMP was weakened after binding.
CONCLUSION: 1) BDMP with procoagulant activity was detected in circulating blood of TBI mice, which may be the material basis of TBI-related coagulation dysfunction; 2) BDMP with high purity was successfully prepared by stepwise centrifugation of brain tissue homogenate in vitro, providing sufficient raw materials for subsequent in vitro experiments; 3) BDMP can activate blood. Platelets, and activated platelets can help BDMP penetrate BBB, which partly explains the mechanism of TBI-related coagulation dysfunction; 4) BDMP can bind to Lactadherin protein and inhibit the coagulation-promoting activity of BDMP. The clearance mechanism of BDMP provides a new idea for the treatment of TBI with coagulation dysfunction.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R651.15

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