Lactadherin蛋白增加TBI小鼠脑源性微粒清除及改善预后的研究

发布时间：2018-04-02 03:02

本文选题：创伤性脑损伤　切入点：Lactadherin　出处：《天津医科大学》2017年博士论文

【摘要】：目的:创伤性脑损伤(traumatic brain injury,TBI)的发病率、致残率和致死率高,而且很难控制原发性损伤,因此控制TBI后的继发性脑损伤成为提高患者预后的重要干预靶点。TBI后继发的凝血功能障碍(traumatic brain injury-associated coagulopathy,TBI-AC)及过度免疫炎症反应是患者伤后致死致残的重要原因,但目前机制尚不明确、临床治疗效果不佳。本课题组前期研究结果显示,TBI后脑源性微粒(brain derived microparticles,BDMP)可大量释放进入循环血,通过激活凝血通路及血小板导致消耗性凝血功能障碍和出血。本次研究将在此基础上,重点研究Lactadherin蛋白能否能增强并加速BDMP的清除,预防或减少TBI-AC的发生,改善TBI的预后及其机制。本研究将丰富对BDMP清除机制的研究,并为治疗TBI-AC及TBI后继发性损伤提供新方法。方法:(1)通过小鼠TBI模型,鼠尾静脉注射Lactadherin观察其是否能降低死亡率并改善TBI的预后。(2)通过伊文思蓝染色观察小鼠TBI模型鼠尾静脉注射Lactadhein能否减少血脑屏障的渗漏。(3)通过磷钨酸-苏木精染色观察小鼠肺组织的纤维素沉积(4)通过ELISA检测血浆中D二聚体的含量变化及通过凝血因子Xa凝血时间实验检测Lactadhein能否减少TBI-AC的发生。(5)通过全血细胞计数研究TBI后血细胞含量的变化。(6)通过流式细胞术检测TBI后血浆中BDMP的含量变化。(7)通过ELISA检测小鼠血浆内Lactadherin蛋白在TBI前后的含量变化。(8)伤后给予Lactadherin蛋白研究使用Lactadherin蛋白治疗的可行性。(9)注射生物素标记的BDMP,并做组织病理染色,明确BDMP的清除途径。(10)通过流式细胞术检测MP,Lactadherin及白细胞复合体的形成。(11)利用Lactadherin基因敲除小鼠及其同窝小鼠验证实验。(12)利用基因蛋白工程技术研究Lactadherin蛋白发挥作用的机制。(13)体外Transwell实验检测Lactadhein能否减少BDMP导致的内皮细胞间的渗漏。(14)体外流式细胞术验证Lactadherin蛋白发挥作用的机制。(15)利用可视流式系统Amnis Image Stream X?Mk II观察mt MP与单核细胞的结合,并尝试阻断其可能的结合位点。结果:(1)注射Lactadherin蛋白能减少TBI小鼠的死亡率,改善预后,降低血脑屏障渗漏,减少肺组织纤维素渗出。(2)体外Transwell实验也证实Lactadherin蛋白能减少内皮细胞间的渗漏(3)注射Lactadherin蛋白能减少TBI小鼠血浆中的BDMP含量,减少血小板的消耗,改善血液的高凝状态。(4)ELISA结果显示TBI后血浆中正常分泌的Lactadherin蛋白含量升高但不足以清除大量释放的BDMP。(5)TBI后给予充足的Lactadherin蛋白能有效地帮助BDMP清除,改善小鼠预后。(6)生物素标记的BDMP定位实验显示Lactadherin蛋白能增加BDMP在肝脏被巨噬细胞清除的量,同时体外实验也能观察到Lactadherin蛋白能促进巨噬细胞粘附吞噬BDMP。(7)通过流式细胞术观察到Lactadherin蛋白主要结合在BDMP的PS上(8)单纯的Lactadherin蛋白的C1C2片段对BDMP的清除能力十分有限,提示Lactadherin蛋白上的EGF片段(该片段能与巨噬细胞上的αvβ3和αvβ5整合素受体结合)是不可缺少的部分。结论:(1)注射Lactadherin蛋白能通过降低血脑屏障渗漏、减少肺组织纤维素渗出、改善血液的高凝状态等几方面减少TBI小鼠的死亡率,改善预后(2)正常分泌的Lactadherin蛋白量较少不足以清除TBI后大量释放的BDMP。(3)Lactadherin蛋白能有效地帮助BDMP与巨噬细胞结合促进吞噬。(4)Lactadherin蛋白的C1C2片段能与BDMP上的PS相结合,EGF片段能与巨噬细胞上的αvβ3和αvβ5整合素结合,要充分的发挥清除作用二者缺一不可。本研究发现了Lactadherin蛋白能减少TBI小鼠的死亡率,改善预后,并揭示了Lactadherin蛋白清除BDMP的机制,为临床治疗TBI后的继发性损伤提供了新的治疗的方法和理论基础。
[Abstract]:Objective: traumatic brain injury (traumatic brain, injury, TBI) incidence rate, disability rate and death rate is high, and it is difficult to control the primary injury, so the control of secondary brain injury after TBI become the important target for intervention.TBI prognosis after coagulation dysfunction secondary to obstruction (traumatic brain injury-associated coagulopathy. TBI-AC) and excessive inflammatory reaction in patients with injury is important reason of death and disability, but the mechanism is not clear, clinical treatment is ineffective. Ourprevious studies showed that TBI brain derived microparticles (brain derived microparticles, BDMP) can be released into the blood circulation, through the activation of the coagulation pathway and lead to platelet consumption coagulation dysfunction and hemorrhage. This study will be based on this, focus on the study of Lactadherin protein can clear can enhance and accelerate the BDMP, prevent or reduce TBI-AC. Students, improve the prognosis and mechanism of TBI. This study will enrich the research on the BDMP removal mechanism, and provide new methods for the treatment of secondary injury after TBI-AC and TBI. Methods: (1) the mouse TBI model, rat tail vein injection of Lactadherin to observe whether it can reduce the mortality and improve the prognosis of TBI (2). Mice TBI model rat tail vein injection of Lactadhein can reduce the leakage of blood brain barrier by Evans blue staining. (3) observation of fibrin deposition in the lung tissue of mice by phosphotungstic acid hematoxylin staining (4) by changing the content of ELISA in plasma and D two dimers and the coagulation factor Xa clotting time assay can Lactadhein to reduce the incidence of TBI-AC. (5) the changes of blood cell content by blood cell count of TBI. (6) by flow cytometry content changes of BDMP in plasma after TBI. (7) detected by ELISA in mouse plasma Lactadh Erin protein content changes before and after TBI. (8) after injury to study the feasibility of the treatment of Lactadherin protein using Lactadherin protein. (9) injection of biotin labeled BDMP and histopathological staining, clear BDMP pathways. (10) detected by MP flow cytometry, the formation of Lactadherin and white blood cell complex. (11) knockout mice and littermate mice experiment using Lactadherin gene. (12) the use of gene engineering technology research of Lactadherin protein play a role in the mechanism. (13) in vitro Transwell assay can reduce Lactadhein leakage in BDMP induced endothelial cells. (14) in vitro flow cytometry verified Lactadherin proteins play a role in the mechanism. (15) the use of video streaming system Amnis Image Stream X? Mk II observation combined with MT MP and mononuclear cells, and try to block its possible binding sites. Results: (1) injection of Lactadherin eggs White can reduce the mortality of TBI mice, improve the prognosis, reduce the blood brain barrier leakage, reduce lung tissue fibrin exudation. (2) Transwell in vitro experiments also confirmed that Lactadherin protein can reduce the leakage between the endothelial cells (3) injection of Lactadherin protein can reduce the content of BDMP TBI in mouse plasma, platelet consumption, hypercoagulable state improve blood. (4) ELISA showed that Lactadherin protein content of normal secretion of TBI in plasma increased but not enough to clear the large release of BDMP. (5) TBI after given sufficient Lactadherin protein can effectively help eliminate BDMP, improve the prognosis of mice. (6) BDMP localization experiments of biotin labeled Lactadherin protein can display the increase of BDMP in the liver was removed and the amount of macrophages, in vitro can also be observed Lactadherin protein can promote the phagocytosis of macrophage adhesion BDMP. (7) Lactad was observed by flow cytometry The herin protein binding to BDMP PS (8) pure Lactadherin protein fragment C1C2 scavenging ability of BDMP is very limited, suggesting that Lactadherin protein on the EGF fragment (with the fragment with macrophages on alpha v beta 3 and alpha v beta 5 integrin receptor) is an indispensable part of the conclusion: (. 1) injection of Lactadherin protein by reducing blood brain barrier leakage, reduce lung tissue fibrin exudation, several aspects of improving blood hypercoagulability and reduce the mortality of TBI mice, improve the prognosis of Lactadherin (2) protein less normal secretion is insufficient to remove TBI after the release of BDMP. (3) Lactadherin protein can effectively help BDMP and macrophage phagocytosis. Combined with the promotion of (4) Lactadherin protein fragment C1C2 can combine with BDMP PS, with EGF fragment and macrophages on alpha v beta 3 and alpha v beta 5 integrin, to fully play the role of the two clear It is indispensable. This study found that Lactadherin protein can reduce the mortality and prognosis of TBI mice, and reveal the mechanism of Lactadherin protein clearance BDMP. It provides a new therapeutic method and theoretical basis for clinical treatment of secondary injury after TBI.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R651.15

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