摘要
第一章解冻的FFP诱导内皮细胞NO分泌机制及功能研究
失血性休克(Hemorrhagic shock, HS)是军民和居民战(创)伤死亡的主要原因。在临床治疗过程中,与传统复苏液乳酸林格氏液(lactated ringer, LR)相比,新鲜冰冻血浆(fresh frozen plasma, FFP)因其在复苏病员中提高患者生存率,具有一定的优势,被临床创伤救治广泛采用。而一氧化氮(nitric oxide, NO)是介导内皮细胞功能重要分子,我们设想FFP复苏失血性休克的疗效可能得益于诱导NO分泌和舒张血管。为了探讨FFP复苏作用分子机制,本文采用NO/Nitrite/Nitrate分析法首先检测了HS大鼠模型FFP及LR复苏后不同时间点血清中NO含量,结果发现FFP复苏后HS大鼠血清中NO含量显著增加(p<0.05)。与单独HS组大鼠相比,FFP复苏组HS大鼠血清中NO含量增加幅度大,出现时间早(p<0.05),而LR复苏却降低大鼠HS复苏后各时间检测点NO含量。同样对体外培养人肺正常微血管内皮细胞(HPMECs)和人真皮淋巴管内皮细胞(HDLECs)FFP处理后NO含量进行分析,结果FFP处理后血管内皮细胞NO生成同样增加,与对照组相比统计学有显著性差异(p<0.05)。为了探讨FFP复苏对HS大鼠血管舒张功能的影响,随后测定各组大鼠乙酰胆碱诱导内皮依赖性血管舒张反应,与单独HS组相比,结果发现FFP复苏显著增加乙酰胆碱诱发内皮依赖性血管舒张反应(分别是49±4mmHg与37±4 mmHg)(p<0.05)。进一步探讨FFP诱导内皮细胞NO分泌分子机制,采用磷酸化蛋白激酶抗体芯片和免疫印迹方法筛选和鉴定FFP处理后内皮细胞相关蛋白激酶磷酸化,结果发现为FFP显著增加两株内皮细胞12种蛋白激酶磷酸化,且在两株内皮细胞中FFP均能诱导AMPKα1,Akt1和eNOS磷酸化。FFP诱导eNOS磷酸化在FFP复苏HS大鼠的肺组织中同样得到了证实。表明FFP体内外均能激活AMPKα1／Akt1／eNOS信号转导通路。分别采用Compound C(AMPK的抑制剂),LY294002(PI3K／Akt抑制剂)或L-NAME(NOS抑制剂)预处理HPMECs细胞,阻挡AMPKα1／Akt1／eNOS信号转导通路,结果为上述三种抑制剂均能抑制FFP诱导eNOS激活和NO生成,提示AMPKα1／Akt1／eNOS信号转导通路激活介导FFP诱导NO分泌和血管舒张。
本项目首次报道FFP诱导增加HS大鼠血清和体外培养内皮细胞上清NO含量,并进一步筛选和鉴定出AMPKα1／Akt1／eNOS信号转导通路介导FFP诱导NO分泌和血管舒张,揭示了FFP复苏保护血管内皮细胞新机制。为了进一步探讨FFP诱导血管内皮细胞增加NO抑制HS诱导血管收缩改善微循环及修复内皮细胞功能提供科学依据。
第二章解冻的FFP对血管内皮细胞迁移的影响及机制研究
失血性休克是创伤后死亡的首要原因。最近的临床研究表明,尽早、尽快地采用新鲜冰冻血浆(fresh frozen plasma, FFP)进行复苏能显著改善临床失血性休克复苏效果。为了满足各地救治中心对FFP日益增长的需求及快速便捷获得FFP,经批准新鲜解冻血浆4℃贮存不多于5天仍可用临床复苏。研究报道FFP中含有高浓度与内皮细胞迁移有关的转化生长因子-β(transform growth factor-β, TGF-β)。由此我们设想FFP可能促进内皮细胞迁移,贮存的FFP可能改变了生长因子水平和信号转导,从而降低FFP疗效。本项目以迁移力作为功能研究着眼点,比较新鲜解冻血浆(FFP Day 0)和4℃贮存5天FFP(FFP Day 5)在正常氧供和缺氧条件下诱导内皮细胞迁移能力及其对内皮细胞TGF-p信号转导通路影响以及FFP贮存过程中TGF-p浓度变化。FFP(Day 0)和FFP(Day 5)分别处理人肺微血管内皮细胞(Human pulmonary microvascular endothelial cells, HPMECs)和人真皮淋巴管内皮细胞(human dermal lymphatic endothelial cells, HDLECs)后,行迁移实验和Western印迹分析,结果发现：1)在正常供氧和缺氧条件下,FFP(Day 0)和FFP(Day 5)均能促进细胞迁移,而FFP(Day 5)促进细胞迁移能力显著下降(p<0.05)；2)FFP(Day 0)与FFP(Day 5)相比,TGF-β1水平显著升高(p<0.05)；3)抑制TGF-βI型受体ALK5的活性后,增强FFP(Day 0)和FFP(Day 5)诱导内皮细胞迁移能力；4)FFP处理后显著增加TGF-p信号转导通路关键分子Smad2／3磷酸化,预处理ALK5活性可抑制其磷酸化。在体外培养两株内皮细胞中,FFP(Day 5)诱导增加Smad2／3磷酸化幅度大,与FFP(Day 0)相比,具有显著性差异(p<0.05)。结果表明TGF-β1／ALK5／Smad2／3信号转导通路参与抑制FFP诱导内皮细胞迁移,标准贮存导致TGF-β1水平升高和增加TGF-β1／ALK5／Smad2／3信号通路转导进而削弱FFP促进内皮细胞迁移。
本项目首次报道FFP不管是在正常供氧还是在缺氧条件下均能诱导内皮细胞迁移,贮存将增加细胞因子TGF-β1水平及增强TGF-β1/ALK5/Smad2/3信号通路转导,并降低FFP诱导内皮细胞迁移,为进一步探讨FFP通过诱导内皮细胞迁移参与失血性休克后血管内皮完整和功能的恢复提供科学依据。
Chapter One:The mechanisms and function of thawed Fresh Frozen Plasma Induces Nitric Oxide Production
Hemorrhagic shock (HS) is the leading cause of death in civilian and military trauma. Fresh frozen plasma (FFP) has been used for HS in trauma centers with survival benefit compared to traditional resuscitation fluids such as lactated Ringer (LR). Because nitric oxide (NO) is a key mediator of normal endothelial function, we hypothesized that part of the beneficial effects of FFP is due to its ability to induce NO production and vasodilation during HS. In a standardized rat HS-resuscitation model, we found that FFP resulted a much higher and earlier NO production in the sera of FFP-resuscitated rats than HS alone rats as determined by a NO/Nitrite/Nitrate assay. On the contrary, LR suppressed HS-induced NO levels at all the time points examined. Acetylcholine induced a significant increase in vasodilation in FFP-resuscitated HS rats compared to HS alone rats (49±4 mmHg vs 37±4 mmHg). In vitro, FFP induced NO production in the conditioned media of FFP-treated human pulmonary microvascular endothelial cells (HPMECs) and dermal lymphatic endothelial cells (HDLECs). Phospho-kinase array and Western blotting revealed that FFP resulted in activation of AMPKα1, Akt1 and eNOS in both HPMECs and HDLECs. eNOS activation was confirmed in the lung tissues of FFP-resuscitated HS rats. Pretreatment with Compound C (AMPK inhibitor), LY294002 (PI3K/Akt inhibitor) or L-NAME (NOS inhibitor) inhibited AMPKα1/Akt1/eNOS cell signaling resulted in FFP-induced NO production and eNOS activation in HPMECs. Together, this study reveals a novel mechanism by which FFP exerts its vasoprotective effect.
Chapter Two:Effect of thawed FFP on Endothelial Cell Migration and its Mechanisms
Hemorrhagic shock (HS) is a leading cause of death after trauma. Recent clinical studies have shown that resuscitation with earlier and increased amounts of fresh frozen plasma (FFP) is associated with improved outcomes after severe HS. Many trauma centers are starting to use thawed plasma, an approved product that can be stored for up to 5 days at 4℃. FFP has been shown to contain significant levels of transforming growth factor-β(TGF-β) which is involved in endothelial cell migration. We hypothesized that FFP promotes endothelial migration, and the storage of FFP may alter its growth factor levels and signaling, thereby leading to decreased efficacy. Using migration as a functional endpoint, we examined the changes of TGF-βsignaling in response to Day 0 versus Day 5 FFP. Human pulmonary microvascular endothelial cells (HPMECs) and human dermal lymphatic endothelial cells (HDLECs) were treated with Day 0 and Day 5 FFPs and were subjected to migration assays and immunoblotting. We found that (1) Day 0 FFP induced endothelial cell migration under both normoxia and hypoxia conditions and this effect diminished significantly in Day 5 FFP; (2) TGF-β1 protein level increased significantly in Day 5 FFP compared to Day 0 FFP; (3) Inhibition of TGF-βtypeⅠreceptor ALK5 enhanced cell migration induced by both Day 0 and Day 5 FFPs; and (4) FFP induced a significant increase in phospho-Smad2/3, which was inhibited by ALK5 inhibitors. Compared to Day 0 FFP, Day 5 FFP induced greater phospho-Smad2/3 in both cell types. Our data suggest that TGF-β/ALK5/Smad2/3 signaling is involved in inhibition of FFP-induced cell migration, and standard storage of FFP may decrease the FFP efficacy on cell migration through increased TGF-P 1 level and TGF-β1/ALK5/Smad2/3 signaling.
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