脑缺血再灌注论文:脑缺血再灌注乌司他丁基质金属蛋白酶9血脑屏障

脑缺血再灌注论文：

脑缺血再灌注 乌司他丁 基质金属蛋白酶

9 血脑屏障 明胶酶谱

【中文摘要】背景与脑梗死是危害人类健康的常见病、多发病,

以病死率、致残率和复发率高为特点,给社会、家庭及个人带来了严

重的经济与精神负担,因此积极寻找有效的治疗措施成为当前研究重

点。为挽救缺血半暗带内损伤较轻的脑组织,及早溶栓重新恢复缺血

组织灌注给脑梗死患者带来了希望,但同时也带来了更严重的再灌注

损伤问题。目前研究认为血脑屏障(BBB)通透性在脑缺血再灌注损伤

的病理生理过程中起重要作用。血脑屏障位于脑实质和其供血血管之

间,由毛细血管内皮细胞及其之间的紧密连接、连续的基底膜和基底

膜外侧的星形胶质细胞终足构成。血管基底膜由细胞外基质分子(ECM)

组成,对维持BBB的完整性起重要作用,而ECM正是基质金属蛋白酶

(MMPs)的主要作用底物。近年来研究发现脑缺血及再灌注后出现MMPs

表达增加,尤其是MMP-9与血脑屏障通透性的增加有关。MMP-9基因

敲除可减小鼠缺血后的脑梗死体积；MMP-9可降解ECM,导致BBB受损

引起出血和水肿,并能促进中性粒细胞浸润,引起局部炎症反应,加重

组织损伤。因此寻找以MMP-9为作用靶点,减轻血脑屏障通透性进而

减轻脑损伤的神经保护剂意义重大。乌司他丁是一种蛋白酶抑制剂,

已有许多研究证实乌司他丁对肝、肾、心、肠、肺等组织缺血再灌注

损伤有器官保护作用。在脑缺血再灌注损伤方面也有少量研究,多侧

重于减轻炎性因子、清除自由基对机体的损伤方面,但对BBB通透性

及MMP-9活性的影响方面还不清楚。本实验采用大脑中动脉线栓法制

备大鼠局灶性脑缺血再灌注损伤模型,检测损伤侧脑组织MMP-9活性

与BBB通透性的变化,并观察乌司他丁治疗后的效果,探讨乌司他丁

对脑缺血的神经保护作用机制,为脑梗死的治疗开辟一条新的途径。

材料与方法健康清洁级SD大鼠72只,体重250g-300g,根据Zea Longa

线栓法并加以改进制作大脑中动脉栓塞再灌注损伤模型。将大鼠随机

分为三组：假手术组(sham,24只),缺血再灌注损伤模型组(MCAO/R,24

只)和乌司他丁处理组(UTI,24只),每组均设4个亚组：6h、24h、48h、

72h,每个亚组6只大鼠。将乌司他丁溶于生理盐水中制成溶液,治疗

组于再灌注后5min内给予腹腔注射乌司他丁(10000U/kg)一次,以后

每天一次。假手术组和模型组给予相应体积生理盐水(1ml/kg)。在各

时间点处死前半小时给予舌下静脉注射2%伊文思蓝

(2%EB,2ml/kg),30min后相应时间点处死大鼠。通过测定损伤侧脑组

织中EB渗出量来观察BBB通透性的改变,用明胶酶谱法检测同侧脑部

MMP-9活性变化。所有数据均以均数±标准差(x±s)表示,采用

SPSS17.0统计软件进行统计学处理。多组数据比较采用单因素方差

(One-Way ANOVA)分析,组间比较采用Bonferroni法,p<0.05有统计

学差异。结果1 EB渗出量：大鼠舌下静脉注射伊文思蓝后,可见大鼠

全身迅速变蓝。通过检测损伤侧脑组织伊文思蓝渗出量,结果表明缺

血再灌注组伊文思蓝渗出量在24h达高峰。乌司他丁处理后EB渗出

量明显降低,结果有统计学意义(p<0.05)。2 MMP-9活性表达：假手

术组脑内MMP-9的活性在各时间点表达均较低；缺血再灌注6h后,

大鼠缺血部位脑组织内可见少量MMP-9的表达,24h明显升高,48h达

高峰,72h明显下降,与假手术组相比有显著性差异(p<0.05)。乌司他

丁处理后脑内MMP-9的表达在6h、24h和48h组升高幅度均较模型组

相同时间点为低,结果有统计学意义(p<0.05)。结论1、大鼠脑缺血-

再灌注后EB渗出量、MMP-9活性表达明显升高,且呈动态变化。说明

BBB通透性、MMP-9参与脑缺血再灌注损伤。2、乌司他丁可能通过降

低MMP-9的活性表达,从而减轻血脑屏障通透性的破坏,这可能是乌

司他丁在脑缺血再灌注损伤的脑保护作用机制之一

【英文摘要】Background and Cerebral infarction,

characterized by high morbidity, mortality, disability and

recurrence rate, as a common disease, has seriously threatened

the health of human being. Because ischemic cerebral diseases

have brought heavy economical and spiritual burden to their

families as well as the whole society, it is important and

urgent to find effective treatment now. Thrombolytic therapy

is proved to be an effective method for some hyperacute cerebral

infarction patients, because it can restore cerebral blood flow

and mitigate ischemic penumbra damage in time. However it also

brings serious reperfusion injuries. Nowadays the role of the

blood-brain barrier (BBB) in the pathogenesis of cerebral

ischemia reperfusion injury becomes more and more

locates between brain tissues and their blood

capillaries. It is consisted of capillary endothelial cells,

their tight junction proteins which bond the endothelial cells

together, multilayered basal lamina and the numerous astrocyte

expansions end feet. In addition the vascular basal lamina is

composed of extracellular matrix proteins (ECM), such as type

IV collagen, laminin, fibronectin, and heparan sulfate, which

plays an important role in maintaining the integrity of BBB

structure. However ECM is the main substrate of matrix

metalloproteinases (MMPs). Recent studies have shown the

increased expression of MMPs after cerebral ischemia

reperfusion, particularly matrix metalloproteinase-9 (MMP-9),

affect the permeability of blood brain barrier greatly. MMP-9

gene knockout of mice are associated with a reduced infarct

volume after cerebral ischemia and reperfusion. MMP-9 not only

degrades the extracellular matrix protein molecules, which

results in a breakdown in the blood-brain barrier and causes

vasogenic edema formation or hemorrhagic transformation, but

also promotes neutrophil infiltration causing local

inflammation response and aggravating tissue damage. So it is

very important to explore proper neuroprotective agents

targeted at y trypsin inhibitor (Ulinastatin, UTI),

extracted and purified from human urine, can inhibit some

serine proteases such as trypsin, chymotrypsin, plasmin, human

leukocyte elastase, and hyaluronidase. Many studies have

confirmed UTI can antagonize ischemia-reperfusion injury in

many other organs such as liver, kidney, heart, intestine, lung,

etc. But few studies have focused on brain, especially the roles

of UTI in inflammatory and scavenging the damage of free

radicals are still unclear, in addition it is still unclear

whether UTI can protect the BBB by influencing the activity of

MMP-9 after cerebral ischemia-reperfusion. We established

reversible middle cerebral artery occlusion (MCAO) with

reperfusion rat model to explore effect of UTI on the

permeability of the blood-brain barrier and the activity of

als and Methods72 clean grade healthy Sprague

Dawley rats, weighting 250-300g, from the Experimental Animal

Center of Zhengzhou University, were randomly divided into

three groups:sham-operated group (sham, n=24), middle cerebral

artery occlusion and reperfusion group (MCAO/R, n=24),

UTI-treated group (UTI, n=24). Each group was further separated

into 6h,24h 48h,72h, four time points, and each time point had

6 rats. The model of focal cerebral ischemia-reperfusion injury

was made by occluding middle cerebral artery and reperfusing

(MCAO/R) referenced from Zea Longa’s with improvement. The

UTI-treated group received an initial intraperitoneal

injection of UTI (10000U/kg) within reperfusion five minutes

and later once a day. The other two groups were delivered

equivalent normal saline (NS,1 ml/kg) by the same way. Evans

blue (2%,2ml/kg weight) was injected through the sublingual

intravenous 30min before they were sacrificed. The BBB

permeability damage was measured by the different degree of

Evans Blue (EB), and the MMP-9 activity was evaluated by gelatin

zymography during a time course ranging from 6 hours to 72 hours

(n=6) of reperfusion following two hours of cerebral ischemia.

All dates were expressed as the mean±standard deviation.

Statistical analyses were handled with SPSS 17.0 software, and

One-way ANOVA and Bonferroni method were taken for data

processing. Significance level was judged by a=s1

the extravasations of EB:Rats turned into blue thoroughly

shortly after Evans blue injection. Damage to the BBB

permeability was judged by extravasations of Evans Blue (EB).

EB extravasations weren’t apparent in the infarction tissue

at 6h, but reached maximum levels at 24h, and afterwards began

to decrease at 48h and 72h. The EB extravasations in the

UTI-treated group decreased significantly compared with the

ischemia/reperfusion (I/R) group (p<0.05).2 the expression of

MMP-9:The levels of MMP-9 were low at all the time points in

sham-operated group. At 6h, a dramatic increase in MMP-9

activity was detected but there was no significant difference

between sham and I/R groups. The activity of MMP-9

significantly elevated at 24h (p<0.05), and reached the peak

at 48h (p<0.05) in I/R group. The MMP-9 activity at 72h (p<0.05)

began decrease, but was still higher than sham-operated group.

The activity of MMP-9 in the UTI-treated 6h,24h 48h, were

significantly reduced compared with the I/R group

(p<0.05).Conclusions1. There were both EB extravasations and

the MMP-9 activity increase in ischemia-reperfusion rats, and

both had dynamic changes. These illustrate that BBB and MMP-9

relates to brain ischemia reperfusion injury.2. UTI can reduce

the permeability of the blood-brain barrier by suppressing the

activity of MMP-9, which may be one of neuroprotective

mechanisms in cerebral ischemia/reperfusion injury.

【关键词】脑缺血再灌注 乌司他丁 基质金属蛋白酶9 血脑屏

障 明胶酶谱

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【说明】本文仅为中国学术文献总库合作提供，无涉版权。作者如有异议请与总库或学校联系。

【英文关键词】Ischemia/reperfusion Ulinastatin

Matrix metalloproteinase-9 Blood-brain barrier

Gelatin zymography

【目录】乌司他丁对脑缺血再灌注损伤大鼠血脑屏障通透性和

MMP-9活性的影响摘要4-7

料和方法15-22

33-36

结果22-23

Abstract7-10

讨论23-32

前言12-15

结论32-33

材

附图

参考文献36-42综述部分 乌司他丁对脑缺血再灌注损

参考文献52-57附录部分伤的保护作用研究进展42-57

57-60缩略词57-58

致谢60

个人简历58-59在学期间发表的学位

论文59-60