摘要
前言
脑卒中是严重威胁人类健康尤其是中老年人健康的常见病，目前对其发病机制的研究正不断深入，但其确切机制尚未完全阐明，还没有令人满意的治疗措施。
预先给予短暂的、亚致死性缺血(即缺血预处理)可以减轻其后发生的严重缺血所造成的组织损伤，此即缺血耐受。脑缺血耐受机制目前还未完全阐明，可能有多种物质参与作用。脑缺血耐受与脑缺血性损伤是有一定联系的。脑缺血预处理与脑缺血损伤在启动机制上很可能有一些相似之处，只是由于刺激程度的不同产生的结果不同。研究证明脑缺血中介导脑损伤的许多物质在脑缺血预处理中却起保护作用。
缓激肽(bradykinin，BK)是外周和中枢神经系统重要的炎症介质，参与脑缺血后的脑损伤。脑缺血后组织和血浆缓激肽水平均升高，缺血后早期应用缓激肽受体拮抗剂明显改善脑缺血大鼠的预后，而在缺血后期转导激肽释放酶基因，可对脑组织产生保护作用。可见缓激肽在脑缺血的不同时程中具有不同的作用。此外，研究证明内源性及外源性缓激肽均对其后的心肌缺血有重要的保护作用。在大鼠小肠微循环实验中也证明，外源性给予缓激肽预处理可抑制缺血后的炎症反应。本实验旨在观察缺血前给予缓激肽预处理，是否会模拟缺血预处理对其后的脑缺血产生保护作用。从而为脑缺血耐受机制的研究及药物预处理的研究提供理论依据。所以本实验首先制备大鼠局灶性脑缺血模型，给予外源性缓激肽预处理，观察其对脑缺血的影响，明确缓激肽预处理的效果和机制，为脑缺血预处理机制的研究提供帮助。
血脑屏障由脑微血管内皮细胞、基膜和胶质细胞足突组成，对于维持脑组织和血液间的物质平衡起重要作用。脑缺血时血脑屏障破坏导致脑血管通透性增加，是缺血性脑损伤与水肿形成病理发展过程中的重要因素。脑毛细血管内皮细胞以紧密连接为特点，缺乏窗孔和胞饮泡。脑缺血后，血脑屏障间紧密连接破坏，胞饮小泡数量增加，白细胞粘附迁移率增加。故本实验进一步采用原代培养的大鼠脑微血管内皮细胞作为体外血脑屏障模型，采用低氧／低糖模拟体内缺血状态，探讨此时血脑屏障结构和功能的改变。此外，研究表明预处理不仅能够保护神经元，而且对内皮细胞也有重要作用。故本实验最后在体外血脑屏障模型中研究预处理时，血脑屏障结构和功能的变化，为进一步研究脑缺血预处理机制提供帮助。
材料与方法
一、缓激肽预处理对大鼠局灶性脑缺血的影响研究
(一)实验材料
1、动物：雄性SD大鼠，体重250-300克。
2、主要试剂：缓激肽，红四氮唑，伊文思兰，兔抗碱性成纤维细胞生长因子(bFGF)多克隆抗体，SABC试剂盒，羊抗兔IgG多克隆抗体，兔抗β-actin多克隆抗体，ECL试剂盒。
3、主要仪器：微量注射泵，分光光度计，电泳仪，转印仪，恒冷冰冻切片机。
(二)实验方法
1、线栓法制备大鼠局灶性脑缺血／再灌注模型。
2、红四氮唑染色和图像分析检测脑缺血梗死体积百分比，分析缓激肽预处理的脑保护效应。
3、干湿重法测定脑缺血后脑水肿程度，分析缓激肽预处理的脑保护机制。
4、伊文思兰染色后，用分光光度计观察血脑屏障通透性的改变，分析缓激肽预处理的脑保护机制。
5、免疫组织化学法测定脑缺血周围组织碱性成纤维细胞生长因子的表达水平，分析缓激肽预处理的脑保护机制。
6、Western-blot法测定脑缺血周围组织碱性成纤维细胞生长因子的表达水平，分析缓激肽预处理的脑保护机制。
二、预处理对脑内皮细胞紧密连接和细胞粘附的影响
(一)实验材料
1、动物：出生3-5d的SD大鼠。
2、主要试剂：DMEM培养基、胎牛血清、ECGS、胰蛋白酶、Ⅱ型胶原酶、葡聚糖、兔抗Ⅷ因子相关抗原抗体、兔抗occludin抗体、鼠抗ZO-1抗体、罗丹明标记鬼笔环肽、兔抗ICAM-1抗体、兔抗VCAM-1抗体、SABC免疫组化试剂盒。
3、主要仪器：二氧化碳培养箱、倒置显微镜、低氧培养箱、荧光显微镜、透射电镜。
(二)实验方法
1、大鼠脑微血管内皮细胞的分离鉴定。
2、采用血气分析仪分析细胞培养液气体含量，确定细胞处于低氧状态。
3、透射电镜观察细胞超微结构，分析氧糖剥夺预处理对脑微血管内皮细胞的保护效应。
4、免疫荧光和荧光标记检测细胞紧密连接相关蛋白ZO-1和细胞骨架蛋白F-actin的位置改变，分析氧糖剥夺预处理对脑微血管内皮细胞的保护机制。
5、免疫组化检测细胞间粘附分子ICAM-1和VCAM-1的表达水平，分析氧糖剥夺预处理对脑微血管内皮细胞的保护机制。
实验结果
一、缓激肽预处理对大鼠局灶性脑缺血的影响研究
1、红四氮唑染色显示缓激肽预处理可明显降低再次缺血时的脑梗死体积，随着缓激肽预处理与脑缺血间隔时间的逐渐缩短，其梗死体积百分比也逐渐减少。
2、缓激肽预处理可明显降低再次缺血时的脑含水量，降低脑水肿程度。
3、缓激肽预处理可使再次缺血时脑组织伊文思兰的渗出量减少，血脑屏障的破坏减轻。
4、免疫组织化学和Western-blot法均表明缓激肽预处理可使再次缺血时缺血周围组织碱性成纤维细胞生长因子的表达增强，从而促进缺血神经元的存活。
二、预处理对脑内皮细胞紧密连接和细胞粘附的影响
1、氧糖剥夺预处理可减轻脑微血管内皮细胞的损伤，降低紧密连接开放程度，对脑微血管内皮细胞有保护效应。
2、氧糖剥夺预处理可使脑微血管内皮细胞紧密连接相关蛋白ZO-1和细胞骨架蛋白F-actin的位置改变减轻。
3、氧糖剥夺预处理可使脑微血管内皮细胞细胞间粘附分子ICAM-1和VCAM-1的表达水平降低。
结论
1、预先给予缓激肽预处理，可对其后的大鼠大脑中动脉缺血再灌注损伤起保护作用，使脑梗死体积减少、脑水肿减轻、血脑屏障通透性降低、脑缺血周围组织碱性成纤维细胞生长因子表达增加。研究证明缓激肽预处理可诱导脑缺血耐受，可能是通过保护脑血管、促进神经元的存活而起作用的。
2、氧糖剥夺／再给氧糖可破坏大鼠脑微血管内皮细胞间紧密连接等超微结构，改变细胞间紧密连接相关蛋白及细胞骨架蛋白的表达，增强细胞间粘附分子的表达；氧糖剥夺预处理可诱导缺血耐受产生，对其后的大鼠脑微血管内皮细胞的损伤起保护作用。
Objective
Stroke is a kind of health-threatening disease especially for the older people. The mechanism of stroke has been studied deeply, but is not fully understood and the treatment measures are not well satisfactory.
Preconditioning with brief, nonlethal ischemia (ischemic preconditioning, IPC) can reduce the injury of subsequent severe ischemia. This phenomenon is called ischemia tolerance. The precise mechanism of brain ischemia tolerance is unclear and a lot of substances may have been involved in the process. Studies revealed there was relationship between brain ischemia tolerance and the injury of cerebral ischemia. The triggering mechanism of brain IPC is similar to that of cerebral ischemia injury, except that different extent of stimuli results in different effects. Studies have illustrated that many substances deteriorating brain damage in ischemia can be protective in the process of brain IPC.
Bradykinin (BK) is considered an important mediator of the inflammatory response in both peripheral and central nervous system, and it has attracted recent interest as a potential mediator of brain injury following stroke. An increase in plasma bradykinin levels was observed during cerebral ischemia. Early treatment after cerebral ischemia with bradykinin B_2 receptor antagonist was reported to improve outcome in ischemic rats. During the late stage of ischemic stroke, kallikrein gene transfer provided neuroprotection against cerebral ischemia injury. These results suggest that during the different period of cerebral ischemia bradykinin acts differently. Studies in heart IPC implicated that not only endogenous bradykinin mediated IPC but also exogenously administrated bradykinin mimicked IPC and protected ischemic myocardium. In the study of rat mesenteric microcirculation, exogenous bradykinin preconditioning resisted inflammatory effects after ischemia. Therefore, we tested bradykinin's ability in a rat middle cerebral artery occlusion model to induce ischemia tolerance against ischemic neuronal injury. It will provide theoretical basis for the studies of brain ischemic tolerance mechanism and drug preconditioning.
Blood-brain barrier (BBB) is composed of brain microvascular endothelial cells, basal membrane and glial cell processes, and which is important to hold the balance between brain tissue and blood. During brain ischemia, the disruption of BBB results in the increase of brain vascular permeability and it could further promote ischemic brain injury and brain edema. Brain microvascular endothelial cells are characterized by the presence of tight junctions and the absence of fenestrations and vesicles. After brain ischemia, the tight junctions of BBB are disrupted, transcytosis vesicles are increased, and leukocytes adherence and migration rate is enhanced. Therefore, we primary culture the rat brain microvascular endothelial cells as BBB model in vitro, take oxygen glucose deprivation (OGD) to mimic ischemia in vivo, and then tested the changes of BBB construction and function. In addition, studies showed that ischemic preconditioning not only could protect neurons but also protect endothelial cells. At last, we tested the influence of preconditioning on BBB construction and function in vitro. It will further provide theoretical basis for the studies of brain ischemic tolerance mechanism.
Materials and Methods
PartⅠ: Effects of bradykinin preconditioning on focal cerebral ischemia rats.
1. Materials
(1) Animals: female SD rats weighing 250-300g.
(2) Experimental reagents: bradykinin, 2,3,5-triphenyltetrazolium, Evans blue, rabbit anti-basic fibroblast growth factor antibody, SABC kit, goat anti-rabbit IgG antibody, rabbit anti-β-actin antibody, enhanced chemiluminescence kit.
(3) Experimental instruments: micro-perfusion pump, spectrophotometer, electrophoresis apparatus, transmark apparatus, freeze microtome.
2. Methods
(1) Rats middle cerebral artery ischemia-reperfusion model by intraluminal suture.
(2) Measurement of infarct volume by 2,3,5-triphenyltetrazolium staining and image analysis to test the effects of bradykinin preconditioning.
(3) Measurement of brain hemispheric water content to demonstrate the mechanism of bradykinin preconditioning.
(4) The blood-brain barrier permeability was quantitatively evaluated by extravasation of Evans blue to demonstrate the mechanism of bradykinin preconditioning.
(5) Immunohistochemical assessment the expression levels of basic fibroblast growth factor surrounding the infarct area to demonstrate the mechanism of bradykinin preconditioning.
(6) Western-blot assessment the expression levels of basic fibroblast growth factor surrounding the infarct area to demonstrate the mechanism of bradykinin preconditioning.
PartⅡ: Effects of preconditioning on brain endothelial cells tight junctions and cell adherence.
1. Materials
(1) Animals: 3-5d SD rats.
(2) Experimental reagents: DMEM, fetal calf serum, ECGS, trypsinase, typeⅡcollagenase, dextran, rabbit anti-Ⅷrelated antigen antibody, rabbit anti-occludin antibody, rat anti-ZO-1 antibody, TRITC-phalloidin, rabbit anti-ICAM-1 antibody, rabbit anti-VCAM-1 antibody, SABC kit.
(3) Experimental instruments: CO_2 incubator, inverted microscope, low-oxygen incubator, fluorescence microscope, electro microscope.
2. Methods
(1) Rat brain microvascular endothelial cells culture and identification.
(2) Analyze the cell culture fluid by blood-gas analyzer to ensure the deoxygenated culture.
(3) Measurements of cell ultrastructure changes by electro microscope to test the effects of OGD preconditioning.
(4) Immunofluorescence and fluorescence-labeled assessment the expression of tight junction proteins ZO-1 and F-actin, to demonstrate the effects of OGD preconditioning on BBB tight junctions.
(5) Immunohistochemical assessment the expression levels of ICAM-1 and VCAM-1 to demonstrate the mechanism of OGD preconditioning.
Results
PartⅠ: Effects of bradykinin preconditioning on focal cerebral ischemia rats.
1. TTC staining results showed that bradykinin preconditioning could reduce infarct volume significantly. And the infarct volume decreased as the time interval between bradykinin administration and ischemic insults shortened.
2. Brain water content in the ischemic hemisphere was significantly reduced and the brain edema was alleviated by bradykinin preconditioning.
3. EB extravasation content and scope of blue-staining were significantly decreased and blood-brain barrier function was protected by bradykinin preconditioning.
4. Both immunohistochemical and Western-blot assessment showed that bradykinin preconditioning significantly increased bFGF-like immunoreactivity surrounding the infarct area and promoted the neuronal survival.
PartⅡ: Effects of preconditioning on brain endothelial cells tight junctions and cell adherence.
1.OGD preconditioning could alleviate the injury of brain microvascular endothelial cells, decrease the opening of tight junctions, protect the brain microvascular endothelial cells.
2. OGD preconditioning could lighten the changes of tight junction proteins ZO-1 and F-actin location.
3. OGD preconditioning could decrease the expression of inter-cellular adherence molecules ICAM-1 and VCAM-1.
Conclusion
1. Bradykinin preconditioning provided neuroprotection against ischemic injury induced by transient middle cerebral artery occlusion in rats. Bradykinin preconditioning decreased infarct volume, brain edema and the permeability of blood-brain barrier and increased the expression of basic fibroblast growth factor surrounding infarct area. The ischemia tolerance in the brain induced by bradykinin preconditioning may be due to the protection of cerebral vasculature and the promotion of neuronal survival.
2. OGD and re-oxygenation/glucose destroyed the ultrastructure such as tight junctions of rat brain microvascular endothelial cells, changed the expression location of cellular tight junctions proteins and cellular framework proteins, increased the expression of inter-cellular adherence molecules. OGD preconditioning induced ischemic tolerance and provided protective effects on rat brain microvascular endothelial cells injury.
引文
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