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低压低氧性肺动脉高压动物模型高凝机制的研究
姚小燕
2016-05-18
导师潘磊
专业内科学
授予单位北京大学
授予地点北京大学第九临床医学院
学位硕士
关键词低压低氧性肺动脉高压 动物模型 高凝状态 TF HIF-1α VEGF
其他题名The study of hypercoagulable state in hypoxia-induced pulmonary hypertension animal model
分类号R543.2
摘要

研究背景:既往研究表明,低压低氧性肺动脉高压在发生血管重构和右心重构的同时,也常常伴随肺动脉原位血栓的形成,但其机制不明。近年来研究表明,缺氧诱导因子-1α(Hypoxia inducible factor-1 alpha,HIF-1α)、血管内皮生长因子(Vascular endothelial growth factor,VEGF)等因子参与了肺动脉高压血管重塑的过程,同时会促使组织因子(Tissue factor,TF)在平滑肌细胞、内皮细胞表达增加。TF是凝血的启动因子,已有研究指出 TF 在冠脉血栓形成、肿瘤高凝状态中均发挥重要作用,但其在肺动脉高压中的研究甚少。

研究目的:本研究旨在通过构建低压低氧性肺动脉高压大鼠模型,探讨TF在肺动脉高压模型的表达情况及其在低压低氧性肺动脉高压高凝状态的潜在机制。

研究方法:健康雄性SD大鼠随机分为常压常氧4周组(对照组),模拟海拔5000米持续低压低氧4周组(实验组)。将实验组大鼠置于全自动调节低压低氧舱(大气压约50kPa,氧浓度10%)内,除每天开舱1小时打扫卫生,补充饮用水、饲料及更换氢氧化钠和氯化钙外,均保持低压低氧环境。4周后采用右心导管检查测定各组大鼠的平均肺动脉压(Mean  pulmonary  arterial pressure,mPAP);用超声仪测定大鼠心率(Heart rate,HR)、右心室游离壁厚度(Right ventricular wall thickness,RVWT);右室舒张末期内径(Right ventricular end diastolic diameter,RVEDD),肺动脉加速时间(Pulmonary artery acceleration time,PAAT),肺动脉射血时间(Pulmonary arterial ejection time,PAET);用电子天平称量右心室(Right ventricula,RV)、左心室(Left ventricular,LV)、室间隔(Interventricular septum,S)重量,以RV/(LV+ S)计右心肥厚指数(Right ventricular hypertrophy index,RVHI)了解右心室肥厚程度;行苏木精-伊红( H-E)染色,观察肺组织显微形态学;采用ELISA法检测血浆中HIF-1α、VEGF、TF的表达。采用免疫组化方法检测肺组织中TF的表达情况。

 

本研究得到北京市科委首都特色项目课题资助,课题编号Z1411070。

 

实验结果:通过低压低氧舱模拟海拔5000米低压低氧环境,持续4周后实验组大鼠的mPAP、RVHI均高于对照组,与对照组比较具有统计学意义(p<0.05);实验组大鼠血浆中HIF-1α、VEGF、游离TF表达升高,与对照组相比具有统计学意义(p<0.05);实验组大鼠肺组织中可见多发动脉血栓形成;实验组大鼠肺血管TF表达升高。

实验结论:

模拟海拔5000米持续低压低氧4周后,检测大鼠的mPAP、RVHI增高,病理可见典型的肺血管重塑和右心重构,低压低氧性肺动脉模型构建成功

超声仪评估大鼠肺动脉高压时,RVEDD、PAET欠稳定,而PAAT、RVWT在评估预测PAH上敏感性和稳定性较好。

血浆、肺组织中TF的表达升高,TF的在内膜表达强阳性,中膜巨噬细胞增多,TF表达强阳性,提示血管内膜、巨噬细胞TF的异常表达与肺血栓形成、高凝状态相关,外源性凝血途径可能在PAH高凝机制中起到重要作用。

英文摘要

Background: Not only pulmonary vascular remodeling and right heart remodeling are important acteristics in pulmonary arterial hypertension, in situ pulmonary thrombosis are often observed. However, the mechanism is still unkown. HIF-1α, VEGF and TF have been involved in vascular remodeling procedure in pulmonary hypertension. Due to the receptor activity for factor VII, TF is primary initiator of the blood coagulation cascade and ensures rapid hemostasis. Inflammatory cytokines, such as HIF-1α, VEGF, can strongly increase the expression of TF in smooth muscle cell and endothelial cells. Recent studies have suggested that plasma TF are positive correlated with coronary artery thrombosis and hypercoagulable state in malignant disease. However, few study is focused on the role of  TF in the hypercoagulability state of pulmonary hypertension.

Objective: This study aims to build hypoxia-induced pulmonary hypertension rat model to investigate the expression and potential role of TF in hypoxia-induced pulmonary hypercoagulable state.

Method: In order to build hypoxia-induced pulmonary hypertension rat model, 18 healthy male SD rats were randomly divided into control group, group of hypobaric hypoxia for 4 weeks simulating 5 km altitude. Rats were sacrificed in 4 weeks and the mPAP (mean Pulmonary arterial pressure) in each rat group were measured by Right-sided heart catheterization. The RV(Right ventricular ), LV(Left ventricular weight), S(interventricular Septum) were weighted by electronic scales, and RVHI (Right heart hypertrophy index) was calculated by RV/(LV + S) to evaluate the degree of right ventricular hypertrophy. Lung tissue microstructure morphology was observed by hematoxylin eosin staining. We used the echocardiography to identify the RVWT(Right ventricular wall thickness), RVEDD(Right ventricular end diastolic diameter), PAAT(Pulmonary artery acceleration time)and PAET(Pulmonary arterial ejection time) of the rat model. The expression plasma HIF-1α, VEGF, TF was analyzed by ELISA. Immunohistochemistry were used to explore the TF expression in the pulmonary vessels.

Results: The average mPAP, RVHI of the experimental group were higher than that of the control group. The PAAT was decreased with the mPAP climbing up in the experimental group. The PAAT in the experimental group was less than control group. The pathological staining suggests that there is an obvious pulmonary arterial remodeling and right heart remodeling in the experimental group. The expression of plasma HIF-1α, VEGF and TF in the experimental group were higher than of the control group. We found that expression of TF was increased in the pulmonary artery in the experimental group.

Conclusion: 1. Using automatic regulating hypobaric hypoxia tank simulating 5km altitude for 4 weeks, the RVSP and the RVHI increase remarkably, the pathological acteristics of rat model are in accordance with hypoxic pulmonary hypertension, which indicated that the hypobaric hypoxia induced-pulmonary hypertension rats model was successfully constructed. 2. In this study, RVWT and PAAT has good correlation in the prediction of right ventricular function by echocardiography. But RVEDD and PAET is not so stable in predictting the right ventricular function. 3. The expression plasma HIF-1α, VEGF, TF is increased in the experimental group compared with the control group. We also found intense TF staining in the vessels of these rats. The increased HIF-1α, VEGF and TF expression in the animal model suggests an association with pulmonary hypertension hypercoagulability state. The extrinsic route of blood coagulation may be related with pulmonary hypertension hypercoagulability.

语种中文
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/124810
专题北京大学第九临床医学院
作者单位北京大学第九临床医学院
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姚小燕. 低压低氧性肺动脉高压动物模型高凝机制的研究[D]. 北京大学第九临床医学院. 北京大学,2016.
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