【作者】 费建文；

【导师】 董亮；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2014， 博士

【摘要】 研究背景肺血栓栓塞症(PTE)是内源性或外源性血栓栓子堵塞肺动脉所引起肺循环障碍的临床和病理生理综合征。PTE发病率、病死率和致残率高,是继恶性肿瘤、心肌梗死之后第三大死亡原因和妇女产后首位死亡原因,已成为国际性医疗保健问题。据文献报道,未治疗的肺栓塞死亡率高达30%,且其PTE漏误诊率极高,欧美国家高达70%,国内则约在90%左右。由于大部分患者得不到及时治疗,急性肺血栓栓塞后大多数患者在解剖、血流动力学方面不能完全恢复正常,另有少数患者发展为预后不良的慢性血栓栓塞性肺动脉高压(CTEPH)。上述情况均伴有肺血管重构,即小的肌型肺动脉中膜增厚和／或非肌型动脉肌型化；其机制主要是在细胞因子作用下,血管平滑肌细胞(SMC)迁移至肺动脉内膜并增殖所致。低分子肝素(LMWH)和尿激酶(UK)均具有抗SMC增殖作用,目前关于LMWH和UK对急性PTE后肺动脉内膜增生作用机制的研究极少。目的1.观察急性肺栓塞后内皮素-1、基质金属蛋白酶-9的变化及不同表达,探讨它们对肺动脉内膜增生的影响机制,并研究其与PTE程度和抗凝溶栓治疗的相关性。2.观察LMWH和UK在急性肺血栓栓塞症后肺动脉新生内膜形成中的作用机制,并进行对比性研究。方法1.雄性SD大鼠124只,随机分为假手术组(Sham组)、肺栓塞组(PTE组)、尿激酶组组(UK组)和低分子肝素组(LMWH组)各30只。制作急性肺栓塞模型过程中死亡4只。鼠尾静脉取血,制备自体血栓；颈静脉插管注入自体血栓栓子制成急性PTE模型。Sham组注入等量生理盐水作对照。LMWH组于PTE模型制成后开始皮下注射LMWH(速碧林,0.01ml/Kg),Q12h,连续7天。UK组于模型制成后开始静脉滴注尿激酶(20000IU/Kg),继以LMWH(速碧林,0.01ml/Kg)皮下注射,Q12h,至7天。Sham组及PTE组皮下注射等量生理盐水,时间同LMWH组。每组分别于栓塞后2h、4h、6h、第1、4、7天各处死5只。2.右心导管法测定肺动脉平均压(mPAP),右心室压力(RVP)。3.右颈动脉插管取血分别进行血气分析、ET-1、MMP-9水平的测定。4.肺组织切片HE染色观察PTE后肺组织病理学特点及肺动脉内膜增生情况。5.采用免疫组织化学方法检测ET-1、MMP-9表达。6. Western blot方法检测肺组织MMP-9的表达。结果1.共94只大鼠注入栓子,术后1小时内死亡3只,术后2小时内死亡1只,2小时后无死亡。所有大鼠注入栓子后均出现呼吸急促、紫绀、心率加快。制成急性PTE模型共90只,成功率95.7%。2.栓塞后PTE、LMWH及UK组mPAP均明显升高。PTE组h2、h4、h6、d1、d4时间点mPAP均升高,与Sham组比较差异有统计学意义(p<0.05)。LMWH组h2、h4、h6、d1时间点mPAP与Sham组比较差异有统计学意义(p<0.05)；与PTE组比较d4时间点差异有统计学意义(p<0.05)。UK组mPAP h2、h4、h6与Sham组比较差异有统计学意义(p<0.05)；与PTE组比较h6、d1、d4有统计学意义(p<0.05)；与LMWH组比较h6、d1差异有统计学意义(p<0.05)。3.栓塞后PTE、LMWH及UK组RVP均明显升高。PTE、LMWH组h2、h4、h6、d1时间点RVP均升高与Sham组比较差异有统计学意义(p<0.05)。UK组RVP h2与Sham组比较差异有统计学意义(p<0.05)；UK组与PTE组、LMWH组比较,h6、d1时间点RVP差异有统计学意义(p<0.05)。4.PTE组所有时间点Pa02均降低与Sham组比较差异有统计学意义(p<0.05)。LMWH组h2、h4、h6、d1、d4PaO2降低与sham组比较差异有统计学意义(p<0.05)；与PTE组比较d4、d,差异有统计学意义(p<0.05)。UK组PaO2h2、 h4、h6、d1与Sham组比较差异有统计学意义(p<0.05)；与PTE组比较h6、d1、d4、d,差异有统计学意义(p<0.05)；与LMWH组比较h6、d1、d4差异有统计学意义(p<0.05)。四组各时间点PaC02比较差异均无统计学意义。5.栓塞后PTE、LMWH及UK组ET-1值均明显升高。PTE、LMWH组h2、h4、h6时间点ET-1值升高与Sham组比较差异有统计学意义(p<0.05)。LMWH组在d4、d,ET-1值明显降低与Sham组比较差异有统计学意义(p<0.05)。UK组h2、h4、h6ET-1值升高与Sham组比较差异有统计学意义(p<0.05)；与PTE组比较h4、h6、d,差异有统计学意义(p<0.05)；与LMWH组比较h4、h。差异有统计学意义(p<0.05)。6.栓塞后PTE、LMWH及UK组MMP-9值均明显升高。PTE组所有时间点MMP-9值与Sham组均有统计学意义(p<0.05)。LMWH、UK组h2、h4、h6、d1MMP-9值与Sham组比较差异均有统计学意义(p<0.05)。LMWH组与PTE组比较d4、d7MMP-9值差异有统计学意义(p<0.05)。UK组与PTE组比较d1、d4、d7MMP-9值差异有统计学意义(p<0.05)。7.免疫组化结果显示：ET-1主要由肺血管内皮细胞、平滑肌细胞、新生内膜中平滑肌细胞、肺泡和支气管上皮细胞等表达。PTE组ET-1呈强阳性表达,LMWH组和UK组均表达弱阳性。MMP-9主要由肺血管内皮细胞、平滑肌细胞、新生内膜中平滑肌细胞及浸润的单核-巨噬细胞表达。栓塞后PTE组MMP-9呈强阳性表达,LMWH组和UK组均呈弱阳性表达。8. Western blot结果显示：PTE、LMWH及UK组MMP-9蛋白表达较Sham组均增高。PTE组MMP-9蛋白表达量明显增高(p<0.05). LMWH和UK两组MMP-9蛋白表达量较PTE组降低,差异有统计学意义(p<0.05),但较Sham组仍高。结论1.经颈静脉注入自体血栓可制成理想的大鼠急性肺栓塞模型。2.大鼠急性PTE后第4天即出现肺动脉内膜增生；肺动脉壁炎性细胞浸润,中膜平滑肌细胞向内膜迁移、增生是急性PTE后肺动脉内膜增生的主要机制；ET-1、MMP-9在大鼠急性PTE后肺动脉内膜增生、肺血管重构过程中起着重要作用。3. LMWH和UK可以减轻大鼠急性PTE后肺动脉内膜增生；还可以降低ET-1、MMP-9血浆中的水平及其在肺动脉中的蛋白合成；UK与LMWH比较,可以更加迅速地缓解呼吸困难、纠正低氧血症、降低肺动脉压力、使栓塞的血管再通。4.急性PTE后肺动脉内膜增生情况比预想要严重的多,这必将影响患者的预后,因此进一步研究急性PTE将对减少CTEPH的发生有至关重要的意义。更多还原

【Abstract】 Background Pulmonary thromboembolism (PTE) is a clinical and physiopathologic syndrom of pulmonary disorder caused by endogenous or exogenous pulmonary thromboembolus. PTE is a disease that of high morbidity, high mortality and high morbidity.It is the third cause of death after malignant tumor and myocardial infarction, and is also the primary death cause in postpartum women. Now PTE has become an international health care. It has been reported that the mortality rate of untreated PTEis as high as30%, while the rate of misdiagnosis and missed diagnosis is extremely high,70%inEurope and the United States, and90%in China. The majority of patients can not get timely treatment, so most of the patients whth acute PTEcan not returned to normalin anatomy and hemodynamics, and a few patients developed into chronic thromboembolic pulmonary hypertension with poor prognosis. Theprocess is accompanied with pulmonary vascular remodeling characterized by medial wall thickness of muscular arteriesand muscularization of peripheral arteries, which may result from increased migration to intima and proliferation of smoothmuscle cells (SMC).Low molecular weight heparin (LMWH) have been shown to inhibit theproliferation of SMC. And urokinase (UK) may have some effects on vascular endothelial cells. At present the research on the mechanism behind the LMWH, UK and pulmonary artery intimal hyperplasia after acute PTE is few.Objective1.To investigate the pathologic characteristics of lung tissus and intimal hyperplasia of pulmonary artery after acute PTE. To study changes of ET-1and MMP-9in lung tissue, and explore their roles in neointima formation associated with acute PTE.2.To investigate the effect of LMWH and UK onintimal hyperplasiaafter acute PTE.Methods 1.124male Sprague-Dawley rats were randomly divided into sham group(n=30), PTE group(n=30),low molecular weight heparin group(LMWH group, n=30) and Urokinase group (UK group, n=30). Experimental pulmonary thromboembolism was induced in94rats by injection of auto-blood clots into jugular vein, and an additional30rats underwent sham operations. after inductionrats received either low molecular weight heparin (LMWH group, LMWH0.01ml/Kg, Q12h), Urokinase group (UK group, UK20000IU/Kg+LMWH0.01ml/Kg, Q12h) or saline (Sham and PTE group)subcutaneously for7days. Animals were sacrificed at2,4,6hours and1,4,7days after PTE. Five animals were killed for each group at each time point.2.Mean pulmonary arterial pressure (mPAP) and right ventricular pressure(RVP) were determined by right heart catheterization.3.Pressure of artery blood oxygen(PaO2), the changes of ET-1and MMP-9were detected by right jugular veinintubation.4. To investigate the pathologic characteristics of lung tissus and intimal hyperplasia of pulmonary artery after acute PTE in lung biopsy by HE staining.5.Immunohistochemical analysis was used to study the changes of ET-1and MMP-9.6. The MMP-9protein content in lung tissue were determined by Western blot.Results1.Induction of PTE was attempted in94rats, with90rats (95.7%) succeeded. Of the4rats died,4died within2hours owing to thrombus retained in the right ventricle of the heart. Increasing of respiratory and heart rate was found in rats after injecting thrombus.2.mPAP in PTE, LMWH and UKgroup were significantly increased.mPAP in PTE group was increased in time points of h2、h4、h6、d1、d4and there were significant differences between PTE and Sham group (p<0.05).There were significant differences in time points of h2、h4、h6、d1between LMWH and Sham group (p<0.05),and there were significant differences in time points ofdl between LMWH and PTE group (p<0.05).There were significant differences in time points of h2、h4、h6between UK and Sham group (p<0.05).There were significant differences in time points of h6、d1、d4between UK and PTE group (p<0.05).There were significant differences in time points of h6、d1between UK and LMWH group (p<0.05). 3.RVP in PTE, LMWH and UKgroup were significantly increased after PTE.RVP in PTE and LMWH group was increased in time points of h2、h4、h6、d1and there were significant differences compared with Sham group (p<0.05).There were significant differences in time point of h2between UK and Sham group (p<0.05).There were significant differences in time point of h6、d1between UK and PTE roup (p<0.05).There were significant differences in time point of h6、d1between UK and LMWH group (p<0.05).4.Pressure of artery blood oxygen(PaO2) in PTE group was decreased in all time points,and there were significant differences compared with Sham group (p<0.05).There were significant differences in time points of h2、h4、h6、d1、d4between LMWH and Sham group (p<0.05).There were significant differences in time point of d4、d7between LMWH and PTE roup (p<0.05).There were significant differences in time point of h2、h4、h6、d1between UK and Sham group (p<0.05).There were significant differences in time point of h6、d1、d4between UK and LMWH group (p<0.05).There were no significant differences between the4groups in PaCO2.5.The level of MMP-9were significantly increased in PTE、LMWH and UK group.There were significant differences in time points of h2、h4、h6between PTE and LMWH group (p<0.05).There were significant differences in time point of d4、 d7between LMWH and Sham roup (p<0.05).There were significant differences in time point of h2、h4、h6between UK and Sham roup (p<0.05).There were significant differences in time point of h4、h6、d7between UK and PTE roup (p<0.05).There were significant differences in time point of h4、h6between UK and LMWH roup (p<0.05).6.The level of MMP-9were significantly increased in PTE、LMWH and UK group.There were significant differences in all the time points between PTE and Sham group (p<0.05).There were significant differences in Sham group in time points of h2、h4、h6、d1compared with PTE and Sham group (p<0.05).There were significant differences in time point of d4、d7between LMWH and PTE roup (p<0.05).There were significant differences in time point of d1、d4、d7between UK and PTE roup (p<0.05).7.Immunohistochemical analysis indicated that ET-1was expressed predominantly in pulmonary vascular smooth muscle cells, macrophages endothelial cells andneointimal smooth muscle cells. ET-1overexpression in PTE group was present on after PTE. ET-1were expressed weakly in LMWH and UK group. MMP-9is expressed in pulmonary vascular smooth muscle cells macrophages, neointimal smooth musclecellsand monocyte-macrophages. The MMP-9expression in PTE group was graduallyenhanced after PTE. MMP-9were expressed weakly in LMWH and UK group.8.In PTE group, MMP-9protein expression increase (P<0.05). In LMWH and UK group, MMP-9protein expression were lower than PTE group (P<0.05), yet the levels of MMP-9protein were higher than those in Sham group.Conclusions1.Rat model of acute pulmonary embolism can be established by injecting autologous blood clots into jugular vein.2.Pulmonary arterial intimal hyperplasia was present at day4and continued to exist after acute PTE in rats.3.ET-1and MMP-9contribute importantly to the process of neointima formation associated with acute PTE.4.LMWH and Urokinase prevents intimal hyperplasia after acute PTE in rats.更多还原