【作者】 张敬霞；

【导师】 王佩显；

【作者基本信息】 天津医科大学 ， 心血管内科， 2002， 博士

【摘要】 背景和目的：急性血栓性肺栓塞是临床常见病，严重威胁着患者的健康和生命。提高本病的治愈率，改善患者的预后，是临床工作者面临的重大问题。临床研究发现，虽经积极的抗凝和溶栓治疗，部分肺栓塞患者肺动脉内的栓子仍未能完全溶解，以至发生慢性血栓栓塞性肺动脉高压，严重降低患者的生活质量乃至危及生命。因此，探讨影响肺动脉内血栓溶解的因素显得尤为重要。研究发现，深部静脉血栓和肺栓塞的栓子本身具有溶栓抵制的特性，主要是基于栓子内PAI-1水平的升高。另一方面，栓塞局部环境内凝血和纤溶因素的变化可能对栓子的溶解也起着一定作用。本研究从凝血系统的启动因素—组织因子(TF)及其抑制物(TFPI)，以及纤溶系统中主要的纤溶酶原激活物(t-PA)及其抑制物(PAI-1)着手，研究栓塞肺动脉组织内各因子mRNA的表达水平，探讨栓塞局部环境凝血和纤溶的变化及该变化对血栓转归的影响，同时研究肺栓塞对全身其它组织凝血和纤溶带来的变化，为临床治疗提供参考。 方法：建立家兔急性血栓性肺栓塞模型，按栓塞时间将其分为栓塞3小时组、8小时组和24小时组，并建立各自的实验对照组和正常对照组。取材肺栓塞家兔的栓塞肺动脉和未栓塞肺动脉(栓塞远端和／或无栓塞的肺动脉分支)，以及实验对照组和正常对照组家兔的正常肺动脉组织，同时取材各组家兔的主动脉、心脏、肝脏和脂肪组织。应用RT-PCR方法检测这些组织TF、TFPI、PAI-1 mRNA表达水平，并用免疫组化方法检测肺栓塞和 天津医科大学 博士研究主学位论文 于常家兔肺动脉、主动脉卜PA和PA－1的抗原表达水平。 实验一．实验性家兔急性血栓性肺栓塞模型建立 结果：1．建立模型成功率高达87．180（34／39），死亡5 ／q。2．每只肺栓塞 家兔肺内可发现2－5个栓子。大多位于肺动脉段和亚段水平。栓塞后3小时 和 8小时，肺动脉内栓子基本完整，未见溶解和破碎；栓塞后24小时，栓 子部分溶解，其中部分血栓被推向肺动脉远端。结论：沿颈静脉插管注入自 体血凝块制作的急性肺栓塞模型成功率较高，操作方便，简单易行，是研究 急性肺栓塞的理想动物模型。 实验二急性血栓性肺栓塞超微病理改变 结果：1．栓塞后3 小时，内皮细胞部分脱落，有极少量蛋臼质凝结；平滑 肌细胞呈合成型，胞浆蟹足状，核仁大，胞核和胞浆暗染，向内皮表面游走 移位；内皮下成片蛋白质沉着，淋巴细胞活化。2．栓塞后S小时，部分内皮 细胞脱落：内皮下水肿，液压增高，可见散在游离红细胞；内皮下淋巳细胞 浸润，可见浓集的多量蛋白质沉积；内膜成纤维细胞功能不良：平滑肌细胞 暗染，呈类凋亡（nara下 ＼ 表浅部平滑肌细胞多呈蟹足状，胞浆 呈合成型；深部平滑肌细胞相互平行，呈梭形收缩型。3．栓塞后24小时，较 严重大片内皮细胞脱落，局部血浆蛋白沉着：内膜下平滑肌细胞移位接近内 工剥脱的课面：内膜下水肿，局部液压增高；部分内膜平滑肌细胞萎缩、变 暗，显示核功能不良，呈类凋亡：内膜基质水肿显著，淋巴细胞活化；内膜 基质肥大细胞活化，在其临近有肥大细胞颗粒抛出、释放，并见个别肥大细 胞颗粒排空呈空囊，核固缩凝集。结论：急性肺栓塞后，栓塞局部肺动脉受 损，组织和结构发生改变，其中平滑肌细胞向内膜迁移，表明损伤血管重构 过程。组织损伤后一些炎性因子以及活化淋巴细胞和肥大细胞释放的部分细 胞因子，在病变的发展中也起着一定作用，推测可能对局部凝血和纤溶产生 影响。 实验三．急性血栓性肺栓塞TF在组织中的表达 结果：1．肺栓塞后 3小时和 8 ／h时，栓塞肺动脉TF mRNA水平显著高于 7 天津医科大学 博士研究生学位论文 未栓塞肺动脉。栓塞肺动脉、未栓塞肺动脉、实验对照组肺动脉TF表达半 定量值，3小时组分别为 1j 土0．ZI，0．40土0．29，0．84土0．26（P（．01）； 8个时组分别为0．84土0．22，0＊5上0．05，0．78土0．38（P（ of）。24小时后 未栓塞肺动脉仆表达开始增强，三组数值分别为0．72土0．55，1．15to．27， 1刀1土0．02，三者比较没有显著性差异（P＞0．05）。2．心脏TF表达在不同栓 塞时间组有统计学差异，栓塞3小时、8小时和24小时组分别为0厂7上0．54， 0．刀士0．卜，0二3土019叩＜0．0引：栓塞8小时和N小时组心脏仆表达亦 低于各自对照组，分别为0。31土O．11，0．52上0．02（P＜0．05）和0．23土0．19， 0．54士0．08 P＜0．05）。3．肺栓塞后主更多还原

【Abstract】 Background and objective: Acute pulmonary embolism (APE) is a very common disease with high morbidity and mortality in clinical practice. Improving the lysis of thrombi in patients with pulmonary embolism remains a challenge. Unfortunately, there are some emboli which can not be lysised completely even under anticoagulation and thrombolytic therapy. Clinical and experimental studies suggest that pulmonary emboli resist endogenous fibrinolysis and lysis induced by plasminogen activator. This resistance to lysis is due in part to specific molecular factors in the thrombus to oppose fibrinolysis. Hence, foundamental insights into the molecular factors that oppose physiological or pharmacological lysis in thrombi will be necessary to improve treatments for pulmonary embolism. Present paper speculates that the lysis of thrombi can also be affected by endogenous coagulation and fibrinolysis system in local circumstances of thromboembolic pulmonary artery after pulmonary embolism. So we analysis the level of some gene expression involving coagulation factor(tissue factor and tissue factor pathway inhibitor) and fibrinolysis factor(tissue plasminogen activator and plasminogen activator inhibitor-I) in the local thromboembolic and non-thromboembolic pulmonary artery in APE rabbit, to find out which factors maybe affect the lysis of emboli in the lung.Methods: Anesthetized 34 rabbits received autologous clots througe jugular vein to establish the pulmonary embolism model. They were divided into groups as follows: APE 3hs, PE 8hs, and PE 24hs group and their corresponding control groups. Thromboembolic, non thromboembolic and normal pulmonary artery were obtained from all groups rabbits. Heart, aorta, liver and adipose tissues alsoobtained from rabbits. All tissues were examined to quantify the amount of TF, TFPI, PAI-1 mRNA expression by RT-PCR method. And also analyzed the expression of PAI-1 and t-PA protein in pulmonary and aortic arteries using immunohistochemical method.Experiment 1. Establishment of rabbit acute pulmonary embolism model Rusults: 1. The successful rate of APE model was 87.18%, 5 of them were died. 2.About 3-5 emboli could be found in the lung of each rabbit, most of them occluded in segment of PA. After APE 3hs and 8hs, the emboli almost remain integrated in the pulmonary artery. While after APE 24 hs, part of emboli had been lysised incompletely, and some of them were put into the distal of pulmonary artery. Conclusion: The successful rate on establishment of APE, which can provide a good model for basic APE study.Experiment 2. Assessment of infrastructure of pulmonary artery in acute pulmonary embolism rabbitResults: 1. APE 3hs group, part of endothelial cell were denudated: smooth muscle cells(SMCs) showed synthesis phenotype, and migrated to endothelium; some protein coagulated beness endothelium; and lymphocyte was activated. 2. APE 8hs group, some of endothelial cell were denudated; subendothelium edema, and fluid pressure was high; large amount of protein were coagulated ; some of SMCs existed para-apoptosis; SMCs in the shallow layer were synthesis phenotype. while in deep layer. SMCs were systolic phenotype. 3. APE 24hs group, a large amount of endothelial cell were denudated; SMCs were migrated to naked faces of endothelium denudated; subendothelium edema, local fluid pressure increased; lymphocyte was activated; and mast cells were also activated. Conclusion: After APE. the local thromboembolic PA were damaged, the tissue and structure also changed, some of cell such as lymphocyte and mast cell were activited. some cytokine secreated from activited cells may affect the coagulationand fibrinolysis in local lesion pulmonary artery.Experiment 3. Expression of tissue factor mRNA in tissues of acute pulmonary embolism rabbitResults: l.APE 3hs and 8hs groups, the level of TF mRNA expression in thromboembolic PA was significantly higher than that of non-thromboembolic PA and normal PA, in 3hs group , they were 1.10±0.21, 0.40±0.29, 0.84±0.26, respective更多还原