调整剪切力抑制内皮细胞多糖包被降解对血管通透性影响的研究

【作者】 公茂磊；

【导师】 刘大为；

【作者基本信息】 北京协和医学院 ， 急诊医学， 2013， 博士

【摘要】 研究目的(1)研究外科大手术后血浆MMPs、多糖包被降解组分的变化及其相关性,研究血浆MMPs、多糖包被降解组分的变化与剪切力的相关性,为干预多糖包被降解提供研究参考。(2)研究剪切力调整对血浆MMPs和多糖包被降解的影响,剪切力与血浆MMPs、多糖包被组分浓度变化的相关性,以及剪切力对肺血管通透性的影响,探讨剪切力干预多糖包被降解改善血管通透性的可能机制,。研究方法实验分为临床试验和动物实验两部分：(1)临床实验分为外科大手术术后组(大手术组)和健康对照组(对照组)2组。入选收住北京协和医院ICU外科大手术术后患者22例和同期健康志愿者18例。大手术组患者入ICU治疗干预前抽取血标本,健康对照组清晨空腹抽取血标本。监测血气分析、血常规、全血粘度、血浆粘度；超声测量右肱动脉内径、Vmax,计算剪切力；分别ELISA法监测血浆MMP-1、MMP-2、TIMP-1、TIMP-3、 CS、HS、syndecan-1浓度。(2)动物实验随机分为：假手术组(sham组)、血液粘度调整组、血流速度调整组3组。血液粘度调整组包括：粘度1组(生理盐水复苏,基线CVP水平)、粘度2组(血浆复苏,基线CVP水平)和粘度3组/速度1组(羟乙基淀粉复苏,基线CVP水平)；血流速度调整组包括：速度1组(羟乙基淀粉复苏,基线CVP水平)、速度2组(羟乙基淀粉复苏,基线值+2mmHg CVP水平)。假手术组(sham组)制备好动物模型后不做任何处理,观察6h。粘度1组：动物放血制备休克模型维持1小时,后予以生理盐水复苏至基线CVP水平,此后以生理盐水持续静脉输入维持CVP于基线水平。粘度2组：动物放血制备休克模型维持1小时,以兔血浆复苏至CVP恢复基线水平,此后以血浆持续静脉输入维持CVP于基线水平。速度1组：动物放血制备休克模型维持1小时,后以羟乙基淀粉复苏至CVP恢复基线水平,此后以羟乙基淀粉持续静脉输入维持CVP于基线水平。速度2组：动物放血制备休克模型维持1小时,以羟乙基淀粉容量复苏至CVP恢复基线水平+2mmHg水平,此后以羟乙基淀粉持续静脉输入维持CVP于此水平。各组均取5只,于复苏后5h每只注射2%伊文思蓝20mg/kg体重,于复苏后7h处死,留取右肺中叶组织标本。各组剩余动物均于复苏后6h处死,留取主动脉标本。各组于动物处死前留取血标本检测血液粘度,超声测定腹主动脉内径、Vmax,计算剪切力；伊文思蓝渗出法测定肺组织伊文思蓝的漏出量；ELISA法监测复苏前和复苏后血浆MMP-1、MMP-2、MMP-7、TIMP-1、TIMP-3、 CS、HS、syndecan-1浓度；ELISA法监测复苏后腹主动脉内皮细胞胞浆中MMP-1、 MMP-2、MMP-7、TIMP-1、TIMP-3、CS、HS、syndecan-1浓度。研究结果(1)临床试验大手术组Hb、Hct、全血粘度、血浆粘度均偏低于对照组。大手术组肱动脉内径、Vmax与对照组无明显差别,但剪切力低于对照组。大手术后MMP-2浓度增加,CS浓度增加,二者成正相关；剪切力与术后血浆MMP-2浓度、CS浓度成负相关。术后MMP-7浓度增加,HS浓度增加,二者成正相关；剪切力与术后血浆MMP-7浓度、HS浓度成负相关。术后TIMP-7浓度降低,syndecan-1浓度增加,二者成负相关。剪切力与术后血浆TIMP-3浓度成正相关、与syndecan-1浓度成负相关。手术后MMP-1浓度升高,但与syndecan-1浓度没有相关性。手术后TIMP-1浓度无明显变化。(2)动物实验采用不同粘度液体复苏,调整血液粘度,成功调整了腹主动脉血流剪切力,用同种粘度液体,维持不同CVP水平,增加SVI,调整血流速度也成功调整了腹主动脉血流剪切力。剪切力水平与反应肺血管通透性的肺组织伊文思蓝(Eevens blue)漏出量成负相关。剪切力与复苏后血浆MMP-7浓度上升幅度、主动脉内皮细胞胞浆MMP-7浓度成负相关；复苏后血浆MMP-7浓度变化与HS浓度变化成正相关；剪切力与复苏后血浆HS浓度上升幅度成负相关、与复苏后主动脉内皮细胞胞浆HS浓度呈正相关。剪切力与复苏后血浆TIMP-3浓度上升幅度、主动脉内皮细胞胞浆TIMP-3浓度成正相关；复苏后血浆TIMP-3浓度变化与syndecan-1浓度变化成负相关；剪切力与复苏后血浆syndecan-1浓度上升幅度成负相关、与复苏后主动脉内皮细胞胞浆syndecan-1浓度呈正相关。剪切力与复苏后血浆MMP-2浓度上升幅度呈负相关,与主动脉内皮细胞胞浆MMP-2呈正相关；复苏后血浆MMP-2浓度变化与CS浓度变化成正相关；剪切力与复苏后血浆CS浓度上升幅度成负相关、与复苏后主动脉内皮细胞胞浆HS浓度呈正相关。研究结论(1)临床试验大手术后患者血浆MMP-2、MMP-7浓度增加,TIMP-3浓度降低,同时大手术后患者血浆多糖包被降解增加。血浆MMP-2、MMP-7、TIMP-3浓度变化分别与CS、HS和syndecan-1浓度变化关系密切。剪切力降低是大手术后血浆CS、HS和syndecan-1脱落的重要影响因素。(2)动物实验采用不同粘度液体复苏或改变心脏前负荷水平增加SVI是调整血流剪切力可行的方法。纠正降低的血流剪切力可改善多糖包被降解。MMP-2、MMP-7和TIMP-3在剪切力改善多糖包被降解过程中发挥重要作用。血流剪切力降低,肺血管通透性增加,而调整剪切力是改善肺血管通透性可行的重要方法。更多还原

【Abstract】 Objective:(1)To investigate changes of concentrations of MMPs and degraded components of glycocalyx in plasma in patients after major surgery, and meanwhile to investigate the correlation between such changes and the correlation between such changes and shear stress in order to provide research references for intervening degradation of glycocalyx after major surgery.(2) To investigate effects of adjustment of shear stress on concentrations of MMPs and degraded components of glycocalyx in plasma, and to investigate the correlation between shear stress and concentrations of MMPs a in plasma or that of degraded components of glycocalyx in plasma respectively, and meanwhile to investigate effects of shear stree on pulmonary vascular permeability in order to explore the possible mechanism of shear stress intervening degradation of glycocalyx and therefore ameliorating of vascular permeability.Methods:The study consisted of one clinical experiment and one animal experiment.(1)Clinical Experiment The experiment totally enrolled18healthy volunteers and22patients after major surgery admitted to the intensive care unit of Peiking union medical college hospital, who were divided into major surgery group and healthy controls respectively. Blood specimens were collected prior to any interventions after admission in major surgery group and blood samples were taken in the morning after fasting for the whole night in healthy controls. Artery gas analysis、blood routine were determined. Blood viscosity and plasma viscosity were determined, and ultrasonic measurement of diameter and Vmax of brachial artery were performed, then shear stress was calculated with the indices mentioned ahead. Concentrations of MMP-1、MMP-2、MMP-7、TIMP-1、TIMP-3、CS、HS、syndecan-1in plasma were also determined by ELISA.(2)Animal Experiment New Zealand Rabbits were randomly divided into three groups, including sham group、blood viscosity adjustment group and blood flow velocity adjustment group. Blood viscosity adjustment group included viscosity group1(resuscitation by saline to baseline CVP level)、 viscosity group2(resuscitation by plasma to baseline CVP level) and viscosity group3/velocity group1(resuscitation by hydroxyethyl starch to baseline CVP level). Blood flow velocity adjustment group consisted of velocity group1(resuscitation by hydroxyethyl starch to baseline CVP level) and velocity group2(resuscitation by hydroxyethyl starch to baseline CVP level+2mmHg). Rabbits were observed for6h after hemorrhage shock animal model was well prepared in sham group. In viscosity group1hemorrhage shock was well prepared by draining blood and maintained for1h, and then followed by resuscitation with saline until CVP returned to the baseline level and such CVP level was maintained by perfusion of saline later on. In viscosity group2hemorrhage shock was well prepared by draining blood and maintained for1h, and then followed by resuscitation with plasma until CVP returned to the baseline level and such CVP level was maintained by perfusion of plasma later on. In velocity group1hemorrhage shock was well prepared by draining blood and maintained for1h, and then followed by resuscitation with hydroxyethyl starch until CVP returned to the baseline level and such CVP level was maintained by perfusion of hydroxyethyl starch later on. In velocity group2hemorrhage shock was well prepared by draining blood and maintained for1h, and then followed by resuscitation with hydroxyethyl starch until CVP returned to the level of the baseline CVP plus2mmHg and such CVP level was maintained by perfusion of hydroxyethyl starch later on. Five rabbits from each group were injected with20mg per kilogram (body weight) of2%evens blue, and sacrificed in the7th hour after resuscitation. Samples of the middle lobe of right lung were taken. The remainders of each group were all sacrificed in the6th hour after resuscitation and Samples of the aorta were taken. Venous blood samples were taken from each animal before sacrifice for measurement of viscosity, which was followed by ultrasonic measurement of diameter and Vmax of aorta, then shear stress was calculated with the indices mentioned ahead for each rabbit. Leakage of evens blues (EB) from lung tissues was measured by EB exudation determination. Concentrations of MMP-1、MMP-2、MMP-7、TIMP-1、TIMP-3、CS、HS、syndecan-1in plasma before and after resuscitation were measured respectively by ELISA for each animal.Concentrations of MMP-1、MMP-2、MMP-7、TIMP-1、TIMP-3、 CS、HS、syndecan-1in endotheliumwere also measured by ELISA for each animal.Results (1) Clinical Experiment Concentrations of Hb、Hct、blood viscosity and plasma viscosity were lower in major surgery group than those in healthy controls. There was no significant difference in the diameter and Vmax of brachial artery between major surgery group and healthy contrls. Concentrations of MMP-2and CS in plasma were increased in major surgery group and positively correlated to each other. The magnitude of shear stress was negatively correlated to both concentration of MMP-2and that of CS in plasma. Concentrations of MMP-7and HS in plasma were increased in major surgery group and positively correlated. The magnitude of shear stress was negatively correlated to both concentration of MMP-7and that of HS in plasma. Concentration of TIMP-3in plasma was decreased in major surgery group and negatively correlated to that of syndecan-1in plasma which was increased after surgery. The magnitude of shear stress was positively correlated to concentration of TIMP-3in plasma and negatively correlated to that of syndecan-1. Concentration of MMP-1in plasma was increased after major surgery, but had no correlation to that of syndecan-1in plasma. Concentration of TIMP-1in plasma was not changed in major surgery group.(2) Animal Experiment Adjustment of blood viscosity by resuscitation with fluids of different viscosity successfully adjusted the magnitude of shear stress in aorta abdominalis, so did adjustment of blood velocity by resuscitation with fluids of the same viscosity to different CVP levels. The magnitude of shear stress was negatively correlated to leakage of EB from lung tissues, which meant elevated magnitude of shear stress could ameliorate pulmonary vascular permeability. The magnitude of shear stress was negatively correlated to both the increase of concentration of MMP-7in plasma and that of MMP-7in aortic endothelium after resuscitation. The increase of concentration of MMP-7in plasma after resuscitation was positively correlated to concentration of HS in plasma after resuscitation. The magnitude of shear stress was negatively correlated to the concentration of HS in plasma and positively to that of HS in aortic endothelium after resuscitation. The magnitude of shear stress was positively correlated to both the increase of concentration of TIMP-3in plasma and that of TIMP-3in aortic endothelium after resuscitation. The increase of concentration of TIMP-3in plasma after resuscitation was negatively correlated to concentration of syndecan-1in plasma after resuscitation. The magnitude of shear stress was negatively correlated to the concentration of syndecan-1in plasma and positively to that of syndecan-1in aortic endothelium after resuscitation. The magnitude of shear stress was negatively correlated to the increase of concentration of MMP-2in plasma and positively to that of MMP-2in aortic endothelium after resuscitation. The increase of concentration of MMP-2in plasma after resuscitation was positively correlated to concentration of CS in plasma after resuscitation. The magnitude of shear stress was negatively correlated to the concentration of CS in plasma and positively to that of CS in aortic endothelium after resuscitation.Conclusion (1) Clinical Experiment Concentrations of MMP-2and MMP-7were increased while that of TIMP-3was decreased after major surgery, meanwhile shedding of CS、HS and syndecan-1were increased respectively. Concentrations of MMP-2、MMP-7and TIMP-3were closely related to those of CS、HS and syndecan-1respectively. Decreased magnitude of shear stress was important factor af fact ing changes of concentrations of CS、HS and syndecan-1in plasma after major surgery.(2) Animal Experiment Resuscitation with fluids of different viscosity and increase of SVI by changing cardiac preload were effective mathods for adjustment magnitude of of shear stress. Adjusted magnitude of shear stress ameliorated degradation of glycocalyx. MMP-2, MMP-7and TIMP-3played a key role in shear stress ameliorating degradation of glycocalyx. Decreased shear stress augmented pulmonary vascular permeability, while adjusted magnitude of shear stress was an feasible and important method for reduction of pulmonary vascular permeability.更多还原