【作者】 白玉鹏；

【导师】 江洪；

【作者基本信息】 武汉大学 ， 内科学， 2013， 博士

【摘要】 第一部分大鼠冠状动脉自体微血栓栓塞模型的构建目的：采用自体微血栓创建一种稳定性好且模拟临床病理生理过过程的大鼠冠脉微栓塞模型。方法：采用健康雄性SD大鼠72只,体重280-320g。随机分为假手术组(SHAM, sham operation group,n=36)、冠脉微栓塞组(CME, Coronary microembolization group,n=36); CME组依注射的血栓剂量为5mg,并根据不同取材时间点再分为术后3小时、24小时和4周3个亚组,每亚组12只。术前尾静脉取血0.5ml,于37℃温箱内形成血凝块,匀浆器研碎血凝块后从主动脉根部内注射注入微血栓制成的混悬液,而微栓塞组注入等量生理盐水。另外96只大鼠(SHAM=48, CME=48)用来进行评价c-TNI水平以及vWF含量以及炎症细胞炎症因子表的的检查,分别于6小时,24小时,1周,4周取材,采用HE、HBFP、CARSTAIRS及Masson染色及电镜观察CME术后3h,4w心肌组织病理学改变；血液动力学、超声心动图检测大鼠心功能变化。结果：与假手术组相比,CME组血浆cTNI以及vWF水平升高,HE、HBFP染色显示CME各组可见不均一的,多个中心小片状心肌缺血、坏死伴炎症细胞浸润增加。CARSTAIRS染色进一步证实心肌微小动脉内(<100μm)有散在的血栓形成。CME各组微梗死面积显著增加,Masson染色显示CME后心肌微小纤维灶形成,且多分布于心内膜区,纤维化增加。超声心动图以及血流动力学监测提示CME组左室收缩功能明显降低。结论：经主动脉根部注射自体微血栓5mg、同时短暂主动脉夹闭可建立稳定、重复性好且模拟人体病变的大鼠冠状动脉微栓塞模型。第二部分大鼠冠状动脉自体微血栓栓塞后微循环障碍及其形成无复流现象的机制研究目的通过构建大鼠自体微栓塞模型探讨微血栓塞后微循环障碍及其形成无复流现象的机制。方法将SD大鼠随机分成自体微栓塞组(CME)和假手术组(SHAM),在血管夹火闭升主动脉时10秒钟从主动脉根部注射0.2ml自体微血栓颗粒,造成冠脉微栓塞模型,假手术组用等量生理盐水注射。模型成功后分别于不同的时间点取大鼠心脏标本。通过注射硫磺素S来评价大鼠心肌的无复流面积,取大鼠血浆测定cTNI以及假性血友病因子(von Willebrand Factor)水平,病理学分析微栓塞后不同直径范围微动脉血管密度的变化,免疫组织化学染色以及免疫印法测定术后炎症因子(IL-6, TNF-a)表达；超声心动图、血液动力学检测大鼠心功能变化。结果与假手术组相比,CME组血浆cTNI以及vWF水平升高,大鼠心肌无复流面积明显增加,免疫组织化学染色提示CME组大鼠直径在10～50μm之间的,尤其是直径在20-50μm之间的的小动脉血管较SHAM组大鼠的数量明显减少,免疫组织化学染色以及免疫印法测定术后炎症因子(IL-6,TNF-a)表达明显增加,且心肌炎症反应不仅局限于微梗灶及周围,同时在许多非梗塞血管也出现炎症细胞明显激活。超声心动图以及血流动力学监测提示CME组左室收缩功能明显降低。结论通过建立大鼠自体冠状动脉微栓塞模型,可以进行冠状动脉微栓塞后微循环障碍及其形成无复流现象的病理生理机制研究。第三部分静脉应用地尔硫卓对大鼠冠状动脉自体微血栓栓塞的治疗作用及其机制的探讨目的：探讨静脉注射地尔硫卓对大鼠冠状动脉自体微血栓栓塞的治疗作用以及可能的机制。材料和方法：在血管夹夹闭大鼠升主动脉10秒钟的同时从主动脉根部用28g的细针注射0.2m1自体微血栓颗粒,造成大鼠的冠状动脉微栓塞模型。造模成功并存活的大鼠被随机分成模型组(CME组,n=38)和地尔硫卓治疗组(CME+DIL组,n=38).地尔硫卓治疗组的大鼠在注射微血栓颗粒5分钟后从尾静脉持续泵入地尔硫卓针剂(1mg/ml,50μg/min/Kg)持续175分钟。血浆cTNI以及vWF水平,血流动力学测定,心脏彩超以及心肌的病理学检查在不同的时间点进行(术后3小时,24小时,7天和28天)。结果：CME组血浆cTNI以及vWF水平升高,HE、HBFP染色显示CME各组可见多中心小灶性心肌缺血、坏死伴白细胞浸润以及炎症因子的表达增加。CARSTAIRS染色进一步证实心肌微小动脉内(<100gm)有散在的血栓形成。CME各组微梗死面积显著增加,无复流面积明显增加,MassFn染色显示CME后心肌微小纤维灶形成,纤维化增加。超声心动图以及血流动力学监测提示CME组左室收缩功能明显降低。与CME组相比,CME+DIL组大鼠在病理学检查上提示心肌损伤以及炎性浸润明显减轻,心功能有所恢复。结论：这种大鼠冠状动脉自体微血栓栓塞模型适合评价静脉注射地尔硫卓的治疗作用,地尔硫卓通过减轻冠状动脉微栓塞以及栓塞后的炎症反应改善了心肌重构和心功能。更多还原

【Abstract】 Part I Establishment and characterization of an Experimental Model of Coronary Thrombotic Microembolism in RatsObjective:To establish a model of coronary thrombotic microembolism in rats.Methods:5mg dried auto-microthrombotic particulates dissolved in0.2ml saline (CM E group) or0.2ml saline (SHAM group) was injected into temporarily clamped aorta of male Sprague-Dawley rats. After auto-microthrombotic particulates injection, serum c-troponin I was measured by electrochemistry and immunofluorescence method and von Willebrand factor (3hours,24hours,1days,28days) was determined by antibody in antigen-based sandwich enzyme-linked immunosorbent assay, myocardial leukocyte infiltration (24hours and7days,28days), percent of arterioles obstructed by thrombosis, early myocardial ischemia or infarct area (3hours),myocardial fibrosis (28days) were observed by HE, CARSTAIRS,HBFP and Masson staining in Light Microscopic Analysis. Cardiac function was evaluated by transthoracic echocardiography and hemodynamic measurements.Results:After automicrothrombotic particulate injection, serum c-troponin I and von Willebrand factor levels, myocardial leukocyte infiltration levels, the percentage of arterioles obstructed by thrombosis, and myocardial fibrosis were all significantly increased whereas cardiac function as evaluated by echocardiography and hemodynamic measurements were significantly reduced compared with the SHAM group.Conclusions:Injection of5mg homologous microthrombotic particle suspension into aorta when clamping the ascending aorta is an effective method to produce coronary microembolism in small animals. Part II The study on microcirculatory disturbance and no-reflow phenomenon mechanism after coronary artery autologous micro thromboembolism in ratsObjective:To research microcirculatory disturbance and no-reflow phenomenon mechanism in the model of coronary thrombotic microembolism in rats.Methods:5mg dried auto-microthrombotic particulates dissolved in0.2ml saline (CME group) was injected into temporarily clamped aorta of male Sprague-Dawley rats. After auto-microthrombotic particulates injection, serum c-troponin I a, von Willebrand factor and ET-level (3hours,24hours,1days,28days) was determined, no-flow area was evaluated by Thioflavin-S (3hours), myocardial leukocyte infiltration (24hours and7days,28days), myocardial expressions of TNFa and IL-6(24hours,1and28days) were measured by Immunohistochemical Analysis and Western Blot Analysis, Arteriole density (AD) was calculated by immunohistochemical analysis. Cardiac function was evaluated by transthoracic echocardiography and hemodynamic measurementsResults:After automicrothrombotic parti cul ate injection, serum c-troponin I and von Willebrand factor levels, the no-flow area as evaluated by Thioflavin S, myocardial leukocyte infiltration levels, myocardial expressions of tumor necrosis factor and interleukin-6, were all significantly increased whereas cardiac function as evaluated by echocardiography and hemodynamic measurements were significantly reduced compared with the SHAM group. Number of arterioles with diameter between10-50μm, especially for arterioles with diameter between20-50μm was significantly lower in CME group at3hours post injection.Conclusion:aortic automicrothrombotic particulate injection could induce coronary microembolism in rats, and this model could be of value in improving the understanding of pathophysiology of no-reflow phenomenon mechanism. Part III Effects of intravenous diltiazem in a rat model of experimental coronary thrombotic microembolismObjective:The objective of this study was to evaluate the feasibility of evaluating the therapeutic effects of intravenous diltiazem in a newly established rat model of coronary thrombotic microembolism (CME).Methods:CME was induced by injecting0.199ml saline containing5mg automicrothrombotic particulates (around10μm) into the aorta of Sprague-Dawley rats over10seconds using a tuberculin syringe with a28-gauge needle. CME rats were randomized to untreated (CME, n=38) group and diltiazem-treated (CME+DIL, n=38) group. Diltiazem (1mg/ml,50μg/min/Kg) was intravenously injected with an infusion pump through tail vein for175minutes at5minutes after automicrothrombotic particulates injection. Hemodynamic measurements, echocardiographic and pathohistogical examinations were performed at various time points (3hours,24hours,7days and28days) post operation.Results:Arteriole thrombosis, multi-focal myocardial necrosis, inflammatory cell infiltration with remarkably increased myocardial TNF-a, IL-6expression and reduced LV systolic function as well as increased plasma vWF, ET-1and c-TNI levels (indicating vascular endothelial injury and myocardial necrosis) were evidenced in CME rats. These pathologic responses in CME rats could be partly attenuated by intravenous diltiazem treatment.Conclusion:This CME model is suitable to evaluate the therapeutic effects of intravenous diltiazem and intravenous diltiazem treatment significantly improves cardiac function through alleviating inflammatory responses and microvascular thrombotic injury in this rat CME model.更多还原