颅内动脉瘤组织超微结构及MMP-9表达相关调控机制的研究

目的 探讨颅内动脉瘤组织超微结构及MMP-9过度表达的相关调控机制.方法 观察颅内动脉瘤标本和正常脑血管的血管壁细胞外基质的形态学变化,检测脑血管壁组织内的CD68、MMP-gmRNA表达水平及c-Jun免疫活性,进行相关统计学分析.结果 电镜下见颅内动脉瘤血管壁的内皮细胞、平滑肌细胞凋亡和细胞外基质(ECM)破坏,CD68在颅内动脉瘤组织内14.60±1.72/高倍视野,正常脑血管壁内未见CD68阳性表达(P<0.01);c-Jun免疫活性在颅内动脉瘤组织内为1.92±051,正常脑血管壁内为0.17±0.41(P<0.01);动脉瘤组织中MMP-9mRNA的表达是正常对照的10.06倍(P<0.01);CD68和MMP-9mRNA之间呈显著正相关(r<,1>=0.931);颅内动脉瘤组织内c-Jun和MMP-9mRNA之间呈显著正相关(r<,2>=0.818).结论 颅内动脉瘤瘤壁组织中阳性表达的CD68可能通过激活c-Jun进而诱导MMP-9的大量表达破坏ECM参与颅内动脉瘤的发病机制.     

c-Jun在颅内动脉瘤中的表达及对MMP-9表达调控作用的研究

目的检测c-Jun在颅内动脉瘤中的表达及探讨其对基质金属蛋白酶-9（MMP-91表达调控的意义。方法对15例颅内动脉瘤标本和6例非脑血管病患者的正常脑血管应用免疫组化SP法和实时荧光定量RT-PCR法分别检测脑血管壁组织内c-Jun免疫活性和MMP-9mRNA的表达水平。结果c-Jun免疫活性在颅内动脉瘤壁组织内为1．92±0．51，正常脑血管壁内为0．17±0．41，二者有显著性差异（P〈0．01）。动脉瘤壁组织中MMP-9mRNA的表达是正常血管的10．06倍（P〈0．01）。颅内动脉瘤壁组织内c-Jun的免疫活性和MMP-9mRNA的表达呈正相关（r=．818，P〈0．001）。结论活化后的c-Jun可能通过其结构域内MMP-9结合位点启动MMP-9大量转录而参与颅内动脉瘤的发病机制。     

炎症反应在颅内动脉瘤发生中作用的研究进展

正颅内动脉瘤是常见的脑血管疾病,发病原因尚不明确。目前认为,颅内动脉瘤发生的机制包括内皮损伤、炎症反应、血管平滑肌表型调节异常、细胞外基质重构、细胞凋亡以及血管壁的缺失。Jamous等[1]发现内皮细胞损伤是动脉瘤管壁最早的改变,紧接着出现基质金属蛋白酶(matrix metalloproteinase,MMP)水解细胞外基质的炎症区域。在动脉瘤发生过程中,白细胞和血管壁平滑肌细胞大量产生     

颅内囊性动脉瘤瘤壁的组织结构变化及其临床意义

目的观察颅内囊性动脉瘤瘤壁超微病理性结构,预测颅内囊性动脉瘤进展。方法在光学显微镜及电子显微镜下观察12例颅内多发动脉瘤（19个标本）、15例单发动脉瘤及9例重型颅脑损伤患者（对照组）脑血管标本的血管壁组织结构。同时采用免疫组化方法检测各组血管壁标本中的Ⅳ型胶原表达情况。结果在动脉瘤标本中可见动脉瘤壁的内皮细胞损伤、弹力板不完整或缺失及平滑肌细胞坏死,与正常大脑中动脉动脉壁有明显的差异;动脉瘤壁Ⅳ型胶原表达较对照组显著减低（P〈0.05）。结论颅内囊性动脉瘤壁中各层结构的形态及分布特点与正常大脑中动脉动脉壁存在较大差异,这可能是颅内囊性动脉瘤容易破裂的原因。     

PDGF-B与IL-1α对大鼠动脉平滑肌细胞MMP-1 mRNA表达影响的实验研究

目的 探讨血小板源件生长因子-B（PDGF-B）与白介素-1α（IL-1α）对动脉平滑肌细胞基质金属蛋白酶（MMP）-1、2及基质金属蛋白酶的组织抑制因子（TIMP）-1表达的影响。方法 通过RT-PCR方法，研究体外原代培养的大鼠主动脉平滑肌细胞在使用PDGF-B及IL-1α的情况下，MMP-1、2及TIMP-1 mRNA表达的情况。结果 正常大鼠动脉平滑肌细胞有MMP-2与TIMP-1 mRNA的表达，但没有MMP-1m RNA的表达，单独使用PDGF-B或IL-1α不能诱导MMP-1的表达，但是同时使用即可诱导其表达。结论 在病理情况下，PDGF-B与IL-1α协同作用诱导MMP-1表达，降解胶原蛋白，推测是颅内动脉瘤生长塑形机制中的重要途径之一。     

iNOS在颅内囊性动脉瘤瘤壁中的表达及意义

目的探讨诱导性一氧化氮合酶（iNOs）在颅内囊性动脉瘤瘤壁中的表达及其意义。方法应用免疫组化染色和原位杂交染色技术检测iNOS在颅内囊性动脉瘤和正常动脉血管壁组织中的表达。结果所有脑动脉瘤标本中均可见iNOS表达，而正常脑动脉标本中则未见其表达：iNOS阳性表达部位主要在动脉瘤壁外膜和中膜的炎症浸润区的炎症细胞（如淋巴细胞、单核巨噬细胞）的胞浆内。结论动脉瘤的发生可能与局部iNOS过度表达，继而合成大量的NO有关。     

实验性大鼠脑动脉瘤形成过程中MMP-2、MMP-9表达的动态变化

目的探讨实验性大鼠动脉瘤形成过程中基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)的表达规律。方法制作肾性高血压大鼠脑动脉瘤模型,通过免疫组化在蛋白水平系统动态观察肾性高血压大鼠脑动脉瘤形成过程中MMP-2、MMP-9表达的变化。结果实验组在术后1 w脑动脉壁即可见MMP-2、MMP-9表达增加,随着术后时间的推移和大鼠血压的增高,其表达也迅速增加,术后1个月基本达最高峰并一直持续至4个月,其中MMP-9较正常状态的增加比MMP-2的表达增加更为显著。对照组脑动脉壁MMP-2、MMP-9也有微弱表达,且MMP-2表达较MMP-9略强。结论脑动脉壁MMP-9、MMP-2特别是MMP-9的过度表达导致的脑动脉壁胶原纤维及内弹力层破坏是脑动脉瘤形成的主要原因之一。     

基质金属蛋白酶-9和血管内皮生长因子在颅内动脉瘤中的表达

目的通过观察基质金属蛋白酶-9(MMP-9)和血管内皮生长因子(VEGF)在颅内动脉瘤中的表达情况,并且与正常脑动脉血管组织中两者的表达相比较,为研究动脉瘤的发病原因及机制提供线索。方法用免疫组织化学方法对16例经手术和病理证实的脑动脉瘤标本中的MMP-9、VEGF的表达进行检测,同时将7例颞叶癫痫间患者手术切除颞极的颞叶皮层动脉血管组织标本作为相对正常脑动脉血管组织的对照,进行MMP-9、VEGF的表达检测,将所得染色标本在光镜下观察,并用医学图像分析系统进行定量分析。结果7例正常脑动脉血管组织中无MMP-9、VEGF阳性表达,而动脉瘤的MMP-9、VEGF阳性表达率分别为81.2%(13/16)和87.5%(14/16),2组标本相对照具有显著差异(P<0.05);MMP9-在动脉瘤壁内、中、外膜中均有表达,其中外、中膜阳性表达较高;VEGF主要在动脉瘤壁中、外膜表达,而内膜表达较少。结论动脉瘤的发生、生长和破裂是个很复杂的过程,MMP-9及VEGF可能是主要的影响因素之一,两者可能与动脉瘤的发生,增长以及结构维持有关。     

人工合成E-选择素对兔颈总动脉动脉瘤壁平滑肌细胞增殖活性的影响

目的 探讨人工合成E-选择素对兔颈总动脉动脉瘤壁平滑肌细胞增殖活性的影响.方法 新西兰白兔54只,雌雄各半,随机分成9组(每组6只),对照组;实验1、2、3、4周组;治疗l、2、3、4周组.用弹性蛋白酶(EA)滴注法建立兔右颈总动脉瘤模型,并用CTA和HE染色观察模型动脉瘤的形态学改变和病理变化,免疫组化分析基质金属蛋白酶-2(MMP-2)、细胞增殖核抗原(PCNA)和平滑肌α肌动蛋白(α-SMA)表达,Real-time PCR检测骨桥蛋白(OPN)和MMP-2 mRNA表达.结果 大体测量和CTA结果显示:治疗组与实验组相比,其动脉瘤高度和宽度均有不同程度减小;免疫组化结果显示:治疗组动脉瘤壁MMP-2蛋白表达与同期实验组相比均有减少,治疗1、2周组动脉瘤壁PCNA蛋白表达低于同期实验组,而治疗3、4周组动脉瘤壁PCNA蛋白表达与同期实验组比较表达升高;治疗组动脉瘤壁α-SMA蛋白表达低于同期实验组.Real-time PCR结果显示:治疗组动脉瘤壁OPN mRNA表达与同期实验组相比均有增加,而治疗组动脉瘤壁MMP-2 mRNA表达分别低于同期实验组.结论 人工合成E-选择素可以抑制兔颈总动脉瘤壁平滑肌细胞数目和层数的减少,有效促进兔颈总动脉瘤壁平滑肌细胞的增殖,使血管平滑肌细胞由收缩型向合成型转化,进而对动脉瘤壁起到一定的修复作用.     

基质金属蛋白酶与实验性自身免疫性脑脊髓炎的免疫屏障

实验性自身免疫性脑脊髓炎（experimental autoimmune cncephalomyelitis，EAE）是一种主要由T细胞介导的，以中枢神经系统（CNSl内小血管周围单个核细胞浸润及白质髓鞘脱失为特征的自身免疫性疾病。EAE炎性脱髓鞘的过程有许多重要的炎性细胞因子参与。基质金属蛋白酶（matrix metalloproteinase，MMPs）尤其是MMP-9、MMP-2是引起血脑屏障（blood brain barrier，BBB）的免疫屏障破坏，炎性细胞浸润以及介导细胞外基质（ECM）和髓磷脂降解的重要因素。[第一段]     

Increased apoptosis and cysteinyl aspartate specific protease-3 gene expression in human intracranial aneurysm.

OBJECTIVE: To investigate apoptosis in vascular smooth muscle cells (VSMCs) and caspase-3 expression in ruptured intracranial aneurysm. METHODS: Tissue samples of 15 ruptured intracranial aneurysms, 6 abdominal aortic aneurysms (AAA) and 6 normal vessels were evaluated. Apoptosis in VSMCs was determined on transmission electron microscopy. Immunohistochemistry for alpha-SMC actin and direct cell counts (medial VSMCs per high-power field (HPF)) were employed to determine medial VSMC density. Additionally, gene expression of caspase-3 was determined using real-time RT-PCR. RESULTS: We demonstrated medial VSMCs exhibiting morphological apoptotic changes in cerebral aneurysm and AAA. Medial VSMC density was significantly decreased in intracranial aneurysm (43.9+/-4.3 SMCs/HPF) and AAA (53.2+/-9.4 SMCs/HPF) compared with the normal arteries (222.8+/-12.1 SMCs/HPF; p<0.01). An 8.94-fold and 6.73-fold increase in expression of caspase-3mRNA in intracranial aneurysm and AAA, respectively, were obtained relative to the normal vessels. CONCLUSIONS: These results suggest that real time RT-PCR provides a useful tool to test gene expression in small samples, and may contribute to a better understanding of the role of apoptosis in ruptured intracranial aneurysm.     

Matrix metalloproteinases 2 and 9 in human atherosclerotic and non-atherosclerotic cerebral aneurysms

Matrix metalloproteinases 2 and 9 (MMP 2 and -9) have been implicated in the pathogenesis of atherosclerosis and aneurysm formation. The goal of the study was to establish the role of these metalloproteinases in both human atherosclerotic and non-atherosclerotic cerebral aneurysms. Eleven cerebral aneurysms (four atherosclerotic, seven non-atherosclerotic) were immunohistochemically stained for MMP 2 and -9. As controls, atherosclerotic and normal Circle of Willis arteries were similarly immunostained. All specimens were retrieved at autopsy and were paraffin-embedded. In order to evaluate the real MMP 2 and -9 activities, gelatin zymography was also performed in only two available specimens of non-atherosclerotic intracranial aneurysms, because of the relative unavailability of fresh intracranial aneurysm tissue (i.e. reluctance to excise the aneurysm fundus at surgery). Our data establish that MMP 2 and -9 were expressed minimally or not at all in normal Circle of Willis arteries but were strongly expressed in medial smooth muscle cells of atherosclerotic Circle of Willis arteries. In the aneurysm group, both MMP 2 and -9 were strongly expressed in the atherosclerotic aneurysms, but MMP 2 alone was detected in the non-atherosclerotic aneurysms. Zymography revealed a weak enzyme activity correlating to MMP 9 standard recombinant protein. MMP 2 activity was not demonstrated in either specimen. This study shows that the expression of MMP 2 and -9 is associated with atherosclerosis, be it in aneurysmal or non-aneurysmal cerebral vessels but MMP 2 appears to be specifically expressed in aneurysms devoid of atherosclerosis perhaps suggesting a pathogenic role for MMP 2 in the alteration of the extracellular matrix of cerebral arteries during aneurysm formation.     

Matrix metalloproteinases MMP-9 and MMP-7 are expressed in experimental autoimmune neuritis and the guillain-barré syndrome

Matrix metalloproteinases (MMPs) are a family of enzymes that may be implicated in the pathogenesis of inflammatory demyelinating disorders such as multiple sclerosis. The present study investigated the expression of 92-kd gelatinase (MMP-9) and five other MMPs in sciatic nerve from Lewis rats with autoimmune experimental neuritis (EAN), an experimental model of the Guillain-Barre syndrome(GBS). Quantitative polymerase chain reaction analysis revealed an up-regulation of MMP-9 mRNA with peak levels concurrent with maximal disease severity. Increased mRNA expression was associated with enhaced enzyme activity, as detected by gelatin zymography. Immunohistochemically, MMP-9 could be localized primarily around blood vessels within the epineurium and enhoneurium in diseased but not normal sciatic nerve. Among all other MMPs investigated, mRNA levels of matrilysin (MMP-7) were found to be up-regulated at the peak of the disorder, remaining at high levels throughout the clinical recovery phase of the disease. To apply these findings to human disease, sural nerve biopsies from GBS patients were examined. By using immunohistochemistry, positive immunoreactivity against MMP-9 and MMP-7 was noted and corroborated by demonstrating augmented mRNA expression in comparison with noninflammatory neuropathies. Furthermore, increased MMP-9 activity was detected by zymography. These findings indicate that 92-kd gelatinase and matrilysin are selectively up-regulated during EAN and expressed in nerves of GBS patients and thus may contribute to the pathogenesis of inflammatory demyelination of the peripheral nervous system.     

血管紧张素Ⅱ和缬沙坦对血管平滑肌细胞增殖和基质金属蛋白酶9表达的影响

目的研究血管紧张素Ⅱ（AngⅡ）对大鼠血管平滑肌细胞（VSMC）的增殖及基质金属蛋白酶-9（MMP-9）表达的影响及缬沙坦（Valsartan）的干预作用。方法分离大鼠胸主动脉平滑肌细胞,贴块法原代培养大鼠血管平滑肌细胞,取第5～10代细胞进行实验,随机分成AngⅡ组（A组）、AngⅡ＋Valsartan组（A＋V组）、Valsartan组（V组）及对照组4组,各组细胞分别采用四唑盐（MTT）比色法检测血管平滑肌细胞增殖,逆转录聚合酶链反应和免疫印迹法测定MMP-9mRNA和蛋白的表达水平。结果与对照组比较,AngⅡ组VSMC的增殖率升高;与AngⅡ组比较,AngⅡ＋Valsartan组VSMC的增殖率降低,差异均明显（P〈0．05）。AngⅡ组与对照组比较,MMP-9mRNA和蛋白的表达均升高;AnⅡValsartan组与AngⅡ组比较,MMP_9mRNA和蛋白的表达均降低,差异均显著（P〈O．05）。结论血管紧张素Ⅱ可以诱导血管平滑肌细胞的增殖和基质金属蛋白酶-9mRNA和蛋白的表达增加;血管紧张素Ⅱ1型受体（AT1R）拮抗剂一缬沙坦可抑制此作用,ATR参与介导此过程。     

NADPH氧化酶对HSV-1感染的小胶质细胞MMP-9激活的影响

目的 探讨NADPH氧化酶在单纯疱疹病毒1型(HSV-1)诱导的小鼠小胶质细胞(BV2)基质金属蛋白酶-9(MMP-9)表达中的作用. 方法 应用0.5、1.0mmol/LNADPH氧化酶特异性抑制剂Apocynin作用HSV-1诱导的BV2细胞,采用明胶酶谱分析细胞培养上清中MMP-9活性;RT-PCR检测NADPH氧化酶亚基p47phox及MMP-9 mRNA的表达;Dihydroethidium(DHE)测定细胞内活性氧簇(ROS)的产生量. 结果 与正常对照组比较,HSV-1诱导的BV2培养上清中MMP-9活性增加,p47phox、MMP-9 mRNA的表达增高,细胞内ROS水平增高约两倍以上,差异均有统计学意义(P＜0.05);0.5 mmol/L Apocynin作用后可使上述变化减轻,但仍高于正常对照组,差异有统计学意义(P＜0.05). 结论 NADPH氧化酶在HSV-1激活小胶质细胞MMP-9表达中可能起重要作用.     

颅内囊性动脉瘤VEGF表达与瘤壁超微结构变化

目的　探讨颅内囊性动脉瘤(ISA)血管内皮细胞生长因子(VEGF)的表达及瘤壁超微结构特征。方法　采用免疫组化方法检测正常脑组织血管和ISA中VEGF的表达情况;透射电镜观察动脉瘤壁超微结构变化。结果　ISA组VEGF表达较正常对照组显著增高(P <0 0 5) ,免疫阳性产物主要分布于动脉瘤壁的中膜层;ISA的内皮细胞变性脱落,胞浆内出现肿胀的线粒体和大小不等的空泡,瘤壁可见形态异常的平滑肌细胞和成纤维细胞,增多的胶原纤维排列紊乱。结论　VEGF表达可能与ISA的血管退行性变以及瘤体的形成、扩大和破裂有重要关系。     

颈动脉内膜切除后基质金属蛋白酶9动态表达对早期血管再狭窄形成的意义

背景：颈动脉内膜切除后发生血管再狭窄是影响治疗效果的重要因素。 目的：观察颈动脉内膜切除后基因基质金属蛋白酶9 mRNA动态表达在早期血管再狭窄发生中的意义。 设计、时间及地点：随机分组设计,对比观察,实验于2006-02/2007-12在解放军北京军区总医院完成。 材料：健康雄性新西兰兔41只,体质量3.0 kg左右,其中36只用于制备颈动脉粥样硬化性狭窄模型。 方法：将41只新西兰兔分为2组,对照组(n=5)：不做任何干预。实验组(n=36)：于造模后4 h,1,3,7,30,90 d 6个时间点各取6只,以40 g/L多聚甲醛灌注固定取材,行常规病理切片苏木精-伊红染色观察形态变化。 主要观察指标：采用实时定量-聚合酶链反应技术观察术前即刻、术后1,3,7 d颈动脉内膜切除后基因基质金属蛋白酶9 mRNA在早期血管再狭窄过程中的表达变化。 结果：颈动脉内膜切除后内膜修复过程大体上可以分为血栓形成阶段,炎症反应阶段,内皮修复阶段,血管平滑肌增殖阶段,基质形成、堆积阶段。基质金属蛋白酶9 mRNA水平在术后1 d开始表达,术后3 d达峰值,术后7 d显著回落。 结论：基质金属蛋白酶9对早期血管平滑肌细胞的增殖、迁移,介导的局部血管重建和再塑,对血管再狭窄的形成有重要作用。     

NF-κB激活及MCP-1和MMP-9表达在兔颈总动脉瘤中的作用

目的 探讨核转录因子(NF-κB)的激活及单核细胞趋化蛋白-1(MCP-1)和基质金属蛋白酶-9(MMP-9)的表达及在脑动脉瘤(CA)形成中的作用.方法 新西兰兔24只,雌雄各半,随机分成4组:A组(对照组)、B组(1周组)、C组(2周组)、D组(3周组),每组6只.用弹性蛋白酶(EA)滴注法建立兔右颈总动脉瘤模型,并用CTA和HE染色观察模型动脉瘤的形态及病理变化,同时用免疫组化分析NF-κB、MCP-1和MMP-9蛋白表达,RT-PCR检测其mRNA的表达.结果 (1)1周后实验兔行CTA检查,18只实验兔均发现作用血管段形成明显的梭形或囊性动脉瘤,其中可见血栓形成,并且血管有闭塞现象,正常对照组6只兔未发现动脉瘤样病变.HE染色检查,实验组动脉瘤瘤壁变薄,弹性纤维断裂,最终消失,平滑肌细胞减少萎缩,降解明显,对照组也有少量炎性细胞浸润,但未观察到弹力纤维降解和平滑肌退行性变.(2)对照组免疫组化显示未见NF-κB及MCP-1表达,MMP-9极少量表达,RT-PCR结果显示均见少量表达;实验组NF-κB及MCP-1均于1周时表达达高峰,3周时表达明显降低,MMP-9表达呈逐渐增强趋势.结论 NF-κB的活化可能是脑动脉瘤发生与发展的始动因素之一,MCP-1及MMP-9表达增高在脑动脉瘤发生发展过程中起着重要作用.

Abstract：

Objective To study the effects of nuclear factor Kappa B( NF - κB) , MCP - 1 and MMP -9 at the early phase of cerebral aneurysm(CA). Methods 24 healthy New Zealand rabbits, half female and half male, were randomly divided into 4 groups: group A (control group), group B( 1 week), group C(2 weeks), group D (3 weeks), 6 rabbits per - group. The morphologic change of the right common carotid artery and formation of the aneurysm were detected by CT angiography and HE dyeing. Aneurysm samples were collected, and immunohistochemistry and real - time rolymerase chain reaction ( Real - time PCR)used to detect the expression of the proteinum and mRNA of NF - κB, MCP - 1 and MMP - 9. Results (1) After a week, using CT angiography to detect all the 18 experimental rabbits, all of them have found visible fusiform or cystiform aneurysms where the elastase was dropped, and also found that some vessels were obstructed. Aneurysms have not found in all the control group rabbits. The pathological examination of the experimental aneurysms showed that the wall of aneurysm becoming thinner, the internal elastic layers were disrupted or disappeared and the reduced medial vascular smooth muscle cell becoming atrophy or degradation obviously. Inflammatory cell infiltrate were also observed in the control group, but the internal elastic layers degradation and vascular smooth muscle degeneration were not found. (2) The result of immunohistochemistry and Real - time PCR indicate that NF - kB and MCP - 1 reached the peak at 1 week, and decreased at 3 weeks. MMP -9 was increased gradually. The expression of NF - kB and MCP -1 were not found in control group. Conclusion NF - κB activation may be one of the initiating agents in the development of the cerebral aneurysm. MCP - 1 and MMP - 9 significantly increased in CA that play an important role in the pathogenesis of cerebral aneurysm (CA).