【作者】 陆国才；

【导师】 苏定冯；

【作者基本信息】 第二军医大学 ， 药理学， 2010， 博士

【摘要】 目的:高血压发病率高,在中国成人高达18.8 %,目前我国大约有2亿高血压病人。脑卒中是高血压病最重要的并发症,近年来脑卒中发生率呈持续上升趋势,且日趋年轻化。脑卒中一旦发生,后果严重,病人非死即残,要使幸存者恢复功能,耗时费力,因此,预防和治疗脑卒中已经成为科学研究迫切需要解决的问题。血压水平是一个非常重要的心脑血管危险因素和预测因子,血压愈高,脑卒中的危险性就愈大,且80 %的脑卒中和52 %的脑梗死因高血压而引发,降低血压可以显著预防脑卒中的发生,但降压治疗并不是唯一的预防策略,并且脑卒中发生的病理生理机制复杂,有必要进行进一步的深入研究,找出除了血压以外的其他关键因子或预防措施,针对这些因子或措施进行干预,作为防治脑卒中的新靶点。近年来,炎症及其潜在治疗靶点成为医学研究的热点。适度的炎症反应有益于宿主抵抗不良因素的侵袭和促进组织修复。但另一方面,短时间内大量的细胞因子的释放及炎症级联反应又可造成严重的病理损伤。几乎在每种心血管疾病的发生和发展过程中,炎症反应均起了一定的作用。目前,动脉粥样硬化已被公认为是一种特异性的炎症反应。脑血管疾病是以血管病变为起始的疾病,与炎症也有着密切的联系。炎症反应参与了缺血性脑卒中的发病及损伤过程。小血管炎症反应是脑缺血后脑损伤级联反应当中的重要一环。脑缺血损伤级联反应中,大量细胞毒性成分诱导自由基和其他介质导致细胞因子和致炎酶原产生炎症反应,细胞因子上调内皮细胞受体,吸引白细胞并诱导他们迁移至受损脑组织。许多报道证实,缺血性脑损害区域内出现白细胞(以中性粒细胞为主)的聚集、浸润,从而加重脑损伤的过程,这一现象受到人们越来越多的关注。而白细胞的聚集、浸润与细胞黏附分子的参与密切相关,其中E-选择素(E-selectin)的作用尤为重要。E-selectin是细胞黏附分子选择素家族的一员,仅表达于活化的血管内皮细胞上。其靶细胞主要是多形核中性粒细胞、单核细胞和淋巴细胞,主要介导血管内皮细胞与淋巴细胞、中性粒细胞及血小板之间的黏附。E-selectin介导的黏附作用既不依赖于白细胞的活化,也不需要白细胞整合素的参与。内皮细胞上的E-selectin可锚定白细胞,有助于白细胞稳定地黏附于内皮细胞,继而迁移至血管外组织。最近的一项临床研究显示,颅内动脉硬化发展为脑卒中与炎症反应密切相关,而血清E-selectin等炎症因子水平可以作为颅内动脉硬化发展为脑卒中的预测因子。另有研究发现,急性脑缺血患者可溶性E-selectin在脑梗死组最高,可逆性脑缺血发作者稍低,短暂性脑缺血发作者最低,说明可溶性E-selectin水平与脑卒中严重程度有关。提示了E-selectin的拮抗剂/活性调节剂治疗急性脑缺血的可能性。由于脑卒中的发病机制复杂,其预防和治疗均有待于深入研究。E-selectin是体内一种重要的细胞黏附因子,但E-selectin是否参与了缺血性脑卒中的发病及损伤过程?E-selectin参与脑卒中发病及损伤的确切机制是什么?目前尚不甚清楚。因此,本课题主要针对这两个问题进行研究,以期发现E-selectin的潜在治疗学意义,为脑卒中的防治研究工作提供新的思路。方法:实验一:利用自发性高血压大鼠(SHR)和脑卒中倾向的自发性高血压大鼠(SHR-SP),比较正常饲养及盐负荷状态下这两种动物血清及脑组织中E-selectin水平的差异。实验二:取C57小鼠和E-selectin基因敲除(Es-/-)小鼠,电凝大脑中动脉(MCA),比较两种动物的脑梗死面积和神经学评分是否有差异。实验三:取C57小鼠和Es-/-小鼠,电凝MCA,应用western blot方法测定两种动物脑组织髓过氧化物酶(MPO)的含量,考察E-selectin对脑缺血后中性粒细胞的浸润是否有影响。实验四:考察E-selectin对脑缺血后炎症反应的影响。比较C57小鼠和Es-/-小鼠脑缺血后脑组织炎症因子IL-6、IL-1β和TNF-α的水平是否有差异。实验五:考察E-selectin对缺血后脑组织细胞凋亡的影响。TUNEL法测定缺血后脑组织的细胞凋亡情况。实验六:电镜检查E-selectin对缺血后脑组织微血管内皮细胞、神经元等细胞形态、超微结构的变化是否有影响。结果:(1)正常饲料组SHR-SP的脑组织和血清中E-selectin的含量均较SHR有升高趋势,但无统计学差异;SHR和SHR-SP喂饲4 %的高盐饲料1个月后,SHR-SP血清和脑组织中E-selectin的含量均显著高于SHR。提示E-selectin有可能参与了SHR-SP自发性脑卒中的发生过程。(2) MCA栓塞后,C57组动物的神经学评分和脑梗死面积均明显低于Es-/-组小鼠,表明E-selectin敲除对脑卒中有显著的保护作用。(3) Western blot结果显示,Es-/-组小鼠脑组织MPO的含量明显低于C57组小鼠,表明E-selectin敲除可以明显减轻缺血后脑组织中性粒细胞的浸润。(4)脑缺血后,脑组织IL-6和TNF-α水平在Es-/-组小鼠和C57组小鼠之间没有明显差异,Es-/-组小鼠的IL-1β水平明显低于C57组小鼠。(5) TUNEL结果显示,Es-/-组小鼠脑梗死区细胞凋亡程度比C57组小鼠明显减轻;表明抑制E-selectin表达可以减轻脑缺血诱导的细胞凋亡。(6)电镜检查结果显示,脑缺血后C57组小鼠的细胞损伤较Es-/-组小鼠严重,C57组小鼠可见核固缩、内皮细胞膜厚薄不均等,而Es-/-组小鼠细胞形态基本正常。结论:(1) E-selectin参与了脑卒中的发生发展过程,抑制E-selectin表达可以减轻急性缺血性脑损伤程度。(2) E-selectin表达受抑制后,脑组织中性粒细胞的浸润明显减轻;脑组织IL-1β的含量显著降低。(3)抑制E-selectin表达可以减轻脑缺血诱导的细胞凋亡及损伤。目的:血管紧张素转换酶抑制剂(angiotensin converting enzyme inhibitors, ACEI)和血管紧张素受体拮抗药(angiotensin receptor blockers, ARB)已经被广泛应用于心脑血管意外的高危人群。目前,ACEI预防脑卒中的有效性得到一致肯定。本研究通过对以往发表的有关ARB治疗心血管意外高危人群的所有随机对照临床试验进行系统评价,以明确ARB预防脑卒中的有效性。方法:电子检索PubMed、EMBASE和Cochrane图书馆为主要来源,搜索至2008年公开发表的ARB干预心脑血管意外高危人群的随机对照临床试验。根据选择标准选择合适的文献,选择标准为比较ARB与安慰剂、ACEI、钙离子拮抗剂预防脑卒中的随机对照临床试验,并对纳入的相关文献进行质量评估和相关数据提取。摘录数据包括患者特征、干预措施、试验质量、治疗终点。主要疗效评判指标为脑卒中。采用RevMan 5.0系统评价软件的随机效应模型,对比较ARB与安慰剂、ACEI、钙离子拮抗剂进行合并,计算效应量,并进行敏感性分析和发表偏倚的评估。结果:总计20项随机对照临床试验纳入荟萃分析,收录108286例患者。安慰剂对照临床试验11项,纳入患者44971例,荟萃分析显示,与安慰剂相比,ARB可以显著降低脑卒中的发生率,合并比值比为0.91 (95 % CI 0.84, 0.98)。总计有6项临床试验(36537例心脑血管意外高危患者)比较了ARB与ACEI预防脑卒中的有效性,荟萃分析的合并比值比为0.93 (95 % CI 0.84, 1.03),两组之间的差异不具有统计学意义。总计有4项临床试验(22446例)比较了ARB与钙离子拮抗剂预防脑卒中的有效性,两组之间的差异同样不具有统计学意义,合并比值比为1.16 (95 % CI 0.91, 1.48)。结论:与安慰剂相比,ARB可以显著降低脑卒中发生率;而与ACEI、钙离子拮抗剂相比,ARB具有相近的脑卒中发生率,因此,相对于高血压、糖尿病、心力衰竭、脑卒中等心脑血管意外高危人群来说,ARB是预防脑卒中的恰当选择。更多还原

【Abstract】 Objectives: Hypertension is one of the most common cardiovascular diseases with high incidence (18.8 %) and mortality resulting from stroke, myocardial infarction, congestive heart failure and end-stage renal disease in China. As the major complication of hypertension, stroke makes a considerable contribution to morbidity and mortality especially for young people. It constitutes a formidable burden of disability and misery for the patients and their relatives and the wider community. Many stroke survivors become dependent, and require lifelong assistance. Given the disease burden of stroke, prevention is an important public health concern to curb the stroke pandemicly. Blood pressure level is one of the most consistent and powerful predictor of stroke. Hypertension accounts for 80 % of stroke risk and 52 % of cerebral infarction attack. So blood pressure control has been conclusively shown to prevent stroke. However, blood pressure level is not the unique determinant for stroke. Here we propose other important determinants for stroke.Accumulating evidence suggests that inflammation plays important roles in the development of acute cerebrovascular disease. Inflammation has been implicated as a secondary mechanism underlying neuronal injury induced by ischemia. A variety of experimental models, including thromboembolic stroke, focal and global ischemia, have been used to evaluate contributions of inflammation to neuronal damage. Endothelial dysfunction represents an early phase of inflammation. Adhesion molecules are involved in leukocyte rolling, firm adhesion and transmigration across endothelial cells, and play an important role in inflammatory disorders. The vasculature endothelium promotes inflammation through upregulation of adhesion molecules that bind to circulating leukocytes and facilitate migration of leukocytes into the central nervous system (CNS). Once being in the CNS, leukocytes produce cytotoxic molecules that promote cell death. During this process, E-selectin is needed to play as an initiator. E-selectin belongs to the selectin family of adhesion molecules and is observed only on activated endothelium. It can serve as an activated endothelial marker. A clinical study indicated that inflammatory factors including E-selectin are involved in the development from intracranial arteriosclerosis to stroke, and serum E-selectin can serve as a predictor. It was found in another study that soluble E-selectin level is highest in patients with cerebral infarction, slight lower in patients with reversible ischemic attack, and lowest in patients with transient ischemic attack, indicating that E-selectin is related to the severity of stroke. These results suggest the promise for the therapy of stroke by using antagonists of E-selectin.As mentioned above, E-selectin is an important adhesion molecule and involved in the onset and pathologic lesion of stroke. But what are the exact mechanisms for the involvement of E-selectin in stroke? Whether E-selectin can be the target for stroke prevention and treatment? In this study, we mainly focussed on these two questions. The effect of E-selectin on stroke and the possible mechanisms involved were investigated. It might develop a new way to find the effective factors involved in the prevention and treatment of stroke. Recent results obtained in clinical trials for asthma and psoriasis show that, although very challenging, the development of E-selectin antagonists holds concrete promise for the therapy of inflammatory diseases.Methods: E-selectin levels of brain and serum were compared between spontaneously hypertensive rats (SHRs) and stroke-prone spontaneously hypertensive rats (SHR-SPs) after a normal diet or a high salt diet (4 % NaCl) was given for 1 month. To further verify the role of E-selectin on stroke, animal model of acute ischemic stroke by electric coagulation of middle cerebral artery were made in E-selectin knockout (Es-/-) mice and C57 mice. The infarct area and hemisphere areas of each section (both sides) were traced and quantified by an image analysis system. Infarct area and neurological score were compared between these two animals. The possible mechanisms of the involvement of E-selectin in stroke were studied in Es-/- mice and C57 mice. The effect of E-selectin on neutrophil infiltration was examined by detecting MPO level in brain using western blot method. To examine the effect of E-selectin on inflammation, levels of IL-6, IL-1βand TNF-αin brain were detected using ELISA method 8 h after MCA occlusion. In situ detection of apoptotic cell by the TUNEL method was performed 24 h after MCA occlusion.Results: The brain and serum E-selectin levels were higher in SHR-SPs than in SHRs (P<0.05) after salt intake, suggesting that E-selectin might be one of the key processes in the pathogenesis of stroke. The ratio of the infarct area and hemisphere areas and neurological score were significantly decreased in Es-/- mice than in C57 mice. MPO level was much lower in Es-/- mice than in C57 mice after MCA occlusion, suggesting neutrophil infiltration was significantly relieved by inhibiting E-selectin expression. IL-1βlevel was significantly higher in C57 mice than in Es-/- mice, but there was no difference in IL-6 and TNF-αlevels between these two animals. Cell apoptosis was significantly lessened in Es-/- mice than in C57 mice.Conclusions: The present study provides evidence that E-selectin is involved in the pathogenesis of stroke. Inhibiting expression of E-selectin presents protective effect on ischemic lesions. This protective effect results from the inhibition of neutrophil infiltration and the reduction of IL-1βlevel, and then relieving the cell apoptosis induced by ischemia. Objectives: Angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) are widely used in patients who are at high risk of cardiovascular events. A consensus has emerged that ACEI can reduce the risk of stroke. This research of overviews of randomized trials was established to investigate the effects of ARB on the prevention of stroke in patients at high risk for cardio-cerebrovascular events.Methods: Electronic databases were searched up to December, 2008, for randomized clinical trials concerning ARB treatments in patients at high risk for cardio-cerebrovascular events. A selection criterion was established to select and evaluate the appropriate literature by comparing randomized trials of ARB with placebo, ACEI, and calcium antagonists. Data were extracted for patients’characteristics, interventions, quality of trials, and rates of stroke. The efficacy measures were the odds ratios of strokes. We did separate overviews of trials comparing ARB with placebo, with ACEI, and with calcium antagonists. The pooled effects were calculated using the random effects model by RevMan 5.0.Results: Twenty randomized clinical trials with 108286 patients were included in the analysis. The overview of placebo-controlled trials (11 trials, 44971 patients) revealed ARBs was associated with a significant reduction in the risk of stroke, with a pooled odds ratio of 0.91 (0.84 to 0.98). In the overview of trials comparing ARB with ACEI (6 trials, 36537 patients), there were no significant reduced risks of stroke with ARB (odds ratio 0.93, 0.84 to 1.03). In the overviews comparing ARB with calcium antagonists (4 trials, 22446 patients), no significant difference was found, with a pooled ratio of 1.16 (0.91 to 1.48).Conclusion: Significant benefits of ARB on the risk of stroke are demonstrated by comparing to the overviews of placebo-controlled trials. There was no evidence of differences when comparing ARB with ACEI, and with calcium antagonists. Therefore, ARB should be regarded as suitable treatments for preventing stroke in patients who are at high risk of cardiovascular events.更多还原