NE对离体脑微血管内皮细胞缺血性损伤的作用
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摘要
背景:缺血性脑血管疾病是人类死亡率最高的三大疾病之一,深入研究缺血性脑血管疾病的病理生理机制,寻找治疗缺血性脑血管疾病的新靶点并开发相应的治疗药物,不仅具有重要的社会效益,也有显著的经济效益。
目前,关于脑缺血/再灌注损伤的病理生理机制研究主要集中在能量代谢障碍、氧自由基应激、细胞内游离钙超载、兴奋性氨基酸释放过多、线粒体的损伤以及神经元细胞凋亡程序启动等。近年来,炎症反应在脑缺血/再灌注中的作用逐渐引起人们的重视。现已证实,中性粒细胞(polymorphonuclear leukocyte,PMN)及炎症细胞因子触发的血管炎症反应在神经元脑缺血/再灌注损伤中具有重要作用。中性粒细胞在脑缺血/再灌注损伤中的作用主要包括两个方面:(1)机械性阻塞作用;(2)释放炎症介质与蛋白水解酶(主要是弹性蛋白酶),加重缺血脑组织损伤。
弹性蛋白酶(neutrophil elastase,NE)是机体内一种具有强烈水解活性的蛋白水解酶,最适底物为弹性蛋白。研究表明,NE不仅参与了多种炎症性疾病如慢性支气管炎、支气管扩张症、生殖道感染等疾病1的发生发展,也在组织、器官的缺血/再灌注损伤中如急性肺损伤起着重要作用,抑制NE活性对这些组织、器官的缺血/再灌注损伤有显著的保护作用。
研究同时发现,大脑微血管内皮细胞受损是脑血管疾病的早期病变和基本诱因。所以,通过研究NE及其特异抑制剂西维来司钠在缺氧复氧过程中对于大脑微血管内皮细胞的影响,则可以进一步证实NE参与了脑缺血/再灌注损伤,NE抑制剂西维来司钠(Sivelestat sodium,Siv)可能对脑缺血/再灌注损伤有保护作用。
目的:(1)进一步探讨大鼠脑微血管内皮细胞的培养方法并对之进行形态学观察。为建立离体血脑屏障提供基础。
(2)建立一种方便、稳定、可靠的体外细胞氧糖剥夺(oxygen glucose deprivation, OGD)/复氧(reoxygenation)模型。(3)明确NE是否通过损伤脑微血管内皮细胞等机制加重缺血/再灌注后的脑损伤,因而NE将成为脑血管疾病治疗一个新的作用靶点,NE抑制剂西维来司钠也将成为一种新型的神经保护剂用于脑血管疾病的治疗,这无疑将会产生巨大的经济效益和社会效益。
方法:原代培养大鼠脑微血管内皮细胞,通过倒置显微镜、免疫细胞化学、电镜进一步鉴定,采用MTT法测定生长曲线,分离大鼠外周血中性粒细胞,建立体外氧糖剥夺/复氧复糖和中性粒细胞损伤模型。将实验分5组:正常对照组(NC组)、缺血/再灌注模型组(I/R组);模型+中性粒细胞损伤组(I/R+PMN组);模型+中性粒细胞损伤+西维来司钠组(I/R+PMN+Siv组),其中,可按西维来司钠的不同浓度分为低、中、高三个剂量亚组;模型+中性粒细胞损伤+依达拉奉组(I/R+PMN+Eda组)。采用ELISA定量测定每组中性粒细胞弹性蛋白酶(NE)的表达、MTT法和LDH测定每组细胞的存活率和损伤程度、放射免疫法测定每组细胞内皮素-1(ET-1)的分泌量、细胞免疫化学定性检测每组细胞间粘附分子-1(ICAM-1)的表达、RT-PCR半定量测定每组BMEC中血管生成素受体-2 mRNA(Tie-2 mRNA)的表达。
结果:(1)经形态学、免疫细胞化学、电镜鉴定所培养的细胞为大鼠脑微血管内皮细胞。
(2)离体BMEC经氧糖剥夺4h/复氧复糖24h后可模拟脑缺血/再灌注损伤。
(3)测定NE、MTT、LDH、ET-1、ICAM-1、Tie-2 mRNA均显示:氧糖剥夺/复氧复糖过程能对BMEC造成损伤,I/R组与NC组比较有明显的统计学差异(p<0.05);中性粒细胞能加重对BMEC的损伤,I/R组与I/R+PMN组之间比较亦有统计学差异(p<0.05);西维来司钠处理后,血管内皮细胞的损伤明显减轻,I/R+PMN组与I/R+PMN+SIR组比较有统计学差异(p<0.05),并且NE测定以及MTT、LDH实验提示在西维来司钠三个剂量组之间存在明显的量-效关系,其对BMEC的保护作用有剂量依赖性。
结论:(1)我们成功的培养出了大鼠微血管内皮细胞,为建立离体血-脑屏障,研究脑血管疾病提供了帮助。
(2)建立了一种方便、稳定、可靠的体外细胞氧糖剥夺/复氧模型。
(3)明确了在脑缺血/再灌注损伤中,NE通过增加ET-1、ICAM-1、Tie-2等物质的表达,损伤脑微血管内皮细胞,进而造成脑微血管的损坏、血脑屏障的破坏,因而NE将成为脑血管疾病治疗一个新的作用靶点,这为NE特异抑制剂西维来司钠作为一种新型的神经保护剂用于脑血管疾病治疗的研究奠定了基础。
Background
Ischemic cerebrovascular disease is a kind of common and frequent disease that severely affects people’s lives and health. It is necessary to study the pathology and physiology mechanism deeply and search the pointed therapy strategy of ischemic cerebrovascular disease. It is important significance for economy and social.
Brain ischemia/reperfusion (I/R) has relationship with many factors, such as free radicals, the excitotoxicity, free calcium over-loading, the dysfunction of mitochondria, inflammation and apoptosis. Recent years, people gradually think highly of the roles of inflammation in brain ischemia/reperfusion injury. It is confirmed that the blood vessel inflammatory reaction by leucocyte (especially polymorphonuclear leukocyte ) and inflammatory cell factors make a important effect in I/R. The effect of polymorphonuclear leukocyte in I/R has two aspect:(1) mechanism blockage (2) releasing mediators of inflammation and proteolytic enzyme(neutrophil elastase) , aggraving the injury of brain tissue.
Neutrophil elastase(NE)is a kind of proteolytic enzyme which has intensive hydrolization and the most optimistic substrate is elastin. Previous study shows that neutrophil elastase either participates many inflammatory disease for example chronic bronchitis, bronchiectasis, gential tract infect or makes a important role in the I/R injury of tissue and organic.
At the same time, it is has proved that the damage of brain microvascular endothelial cells (BMEC) is the earlier pathological changes and basic remote cause in ischemic cerebrovascular disease. Therefore, we can further confirm that NE participate the ischemia /reperfusion injury,and the NE inhibitor Sivelestat Sodium likely to cerebral ischemia / reperfusion injury protection through the study of BMEC in oxygen glucose deprivation/reoxygenation.
Objective
(1) To isolate and to observe the morphology of brain microvascular endothelial cells from Wistar rat.
(2) To establish a simple, stable and reliable model of oxygen glucose deprivation/reoxygenation (OGD/R) of microvascular endothelial cells in vitro.
(3) To investigate whether NE aggtavate I/R injury though impairing brain blood capillary. So NE will become the new pointed therapy strategy in the therapy of ischemic cerebrovascular disease. And the Sivelestat sodium will be a new nerves protective additive in the therapy of cerebrovascular disease.
Methods
BMEC of 5～7 days Wistar rats were primary cultured. The BMEC were affirmed by immunocytochemical method, morphologies under light microscope and ultramicrostructure under electron microscope. We observed the growth curve of BMEC by MTT assay. We established a model of oxygen glucose deprivation/reoxygenation (OGD/R) of microvascular endothelial cells in vitro. The experiment has been divided 5 groups: normal control group (NC group); ischemia/reperfusion group (I/R group); ischemia/reperfusion + polymorphonuclear leukocyte group (I/R+PMN group); ischemia/reperfusion + polymorphonuclear leukocyte + Sivelestat sodium group (I/R+PMN+Siv group); polymorphonuclear leukocyte + Edaravone group (I/R+PMN+EDA group). We quantitative assayed the NE by ELASA; estimate cells survival and degree of injury by MTT and LDH; assayed the endothelin-1 secretory volume by radioactive immunoassay (RIA); detected the intercellular adhesion molecule-1 (ICAM-1) by Immuneeocytochemical method ,and assayed the angiogenin receptor-2 mRNA (Tie-2 mRNA) by RT-PCR.
Results
(1) The characterization of BMEC was confirmed based on the morphology and positive by the detection of factorⅧantigen.
(2) Oxygen glucose deprivation for 4 hours and reoxygenation for 24 hours can simulate the ischemia/reperfusion injury of BMEC in vivo.
(3)NE、MTT、LDH、ET-1、ICAM-1 and Tie-2experiment show: in the process of oxygen glucose deprivation/reoxygenation ,BMEC have subjected great damagement, I/R groups have significant differences compared with NC groups (p<0.05);polymorphonuclear leukocyte can aggregrate this injury, I/R+PMN groups have significant differences compared with I/R groups (p<0.05); after Sivelestat Sodium treatment, the effect of I/R injury has been decreased, I/R+PMN+Siv groups have significant differences compared with I/R+PMN groups (p<0.05), and NE、MTT、LDH experiment show that the three dose groups there was a significant dose-effect relationship. The protective effect on the BMEC has a dose-dependent manner.
Conlcusions
(1) We isolated and cultured BMEC in vitro successfully. The methods provided a useful way to study the cerebrovascular disease in vitro.
(2) We have established a simple, stable and reliable model of I/R of vascular endothelial cells in vitro successfully.
(3) It is confirmed that NE injury brain blood capillary and aggravate I/R brain injured through increasing the expression of ET-1、ICAM-1、Tie-2. So NE will become the new pointed therapy strategy in the therapy of ischemic cerebrovascular disease. And this will lay a foundation for the Sivelestat sodium as a new nerves protective additive in the therapy of cerebrovascular disease.
目前,关于脑缺血/再灌注损伤的病理生理机制研究主要集中在能量代谢障碍、氧自由基应激、细胞内游离钙超载、兴奋性氨基酸释放过多、线粒体的损伤以及神经元细胞凋亡程序启动等。近年来,炎症反应在脑缺血/再灌注中的作用逐渐引起人们的重视。现已证实,中性粒细胞(polymorphonuclear leukocyte,PMN)及炎症细胞因子触发的血管炎症反应在神经元脑缺血/再灌注损伤中具有重要作用。中性粒细胞在脑缺血/再灌注损伤中的作用主要包括两个方面:(1)机械性阻塞作用;(2)释放炎症介质与蛋白水解酶(主要是弹性蛋白酶),加重缺血脑组织损伤。
弹性蛋白酶(neutrophil elastase,NE)是机体内一种具有强烈水解活性的蛋白水解酶,最适底物为弹性蛋白。研究表明,NE不仅参与了多种炎症性疾病如慢性支气管炎、支气管扩张症、生殖道感染等疾病1的发生发展,也在组织、器官的缺血/再灌注损伤中如急性肺损伤起着重要作用,抑制NE活性对这些组织、器官的缺血/再灌注损伤有显著的保护作用。
研究同时发现,大脑微血管内皮细胞受损是脑血管疾病的早期病变和基本诱因。所以,通过研究NE及其特异抑制剂西维来司钠在缺氧复氧过程中对于大脑微血管内皮细胞的影响,则可以进一步证实NE参与了脑缺血/再灌注损伤,NE抑制剂西维来司钠(Sivelestat sodium,Siv)可能对脑缺血/再灌注损伤有保护作用。
目的:(1)进一步探讨大鼠脑微血管内皮细胞的培养方法并对之进行形态学观察。为建立离体血脑屏障提供基础。
(2)建立一种方便、稳定、可靠的体外细胞氧糖剥夺(oxygen glucose deprivation, OGD)/复氧(reoxygenation)模型。(3)明确NE是否通过损伤脑微血管内皮细胞等机制加重缺血/再灌注后的脑损伤,因而NE将成为脑血管疾病治疗一个新的作用靶点,NE抑制剂西维来司钠也将成为一种新型的神经保护剂用于脑血管疾病的治疗,这无疑将会产生巨大的经济效益和社会效益。
方法:原代培养大鼠脑微血管内皮细胞,通过倒置显微镜、免疫细胞化学、电镜进一步鉴定,采用MTT法测定生长曲线,分离大鼠外周血中性粒细胞,建立体外氧糖剥夺/复氧复糖和中性粒细胞损伤模型。将实验分5组:正常对照组(NC组)、缺血/再灌注模型组(I/R组);模型+中性粒细胞损伤组(I/R+PMN组);模型+中性粒细胞损伤+西维来司钠组(I/R+PMN+Siv组),其中,可按西维来司钠的不同浓度分为低、中、高三个剂量亚组;模型+中性粒细胞损伤+依达拉奉组(I/R+PMN+Eda组)。采用ELISA定量测定每组中性粒细胞弹性蛋白酶(NE)的表达、MTT法和LDH测定每组细胞的存活率和损伤程度、放射免疫法测定每组细胞内皮素-1(ET-1)的分泌量、细胞免疫化学定性检测每组细胞间粘附分子-1(ICAM-1)的表达、RT-PCR半定量测定每组BMEC中血管生成素受体-2 mRNA(Tie-2 mRNA)的表达。
结果:(1)经形态学、免疫细胞化学、电镜鉴定所培养的细胞为大鼠脑微血管内皮细胞。
(2)离体BMEC经氧糖剥夺4h/复氧复糖24h后可模拟脑缺血/再灌注损伤。
(3)测定NE、MTT、LDH、ET-1、ICAM-1、Tie-2 mRNA均显示:氧糖剥夺/复氧复糖过程能对BMEC造成损伤,I/R组与NC组比较有明显的统计学差异(p<0.05);中性粒细胞能加重对BMEC的损伤,I/R组与I/R+PMN组之间比较亦有统计学差异(p<0.05);西维来司钠处理后,血管内皮细胞的损伤明显减轻,I/R+PMN组与I/R+PMN+SIR组比较有统计学差异(p<0.05),并且NE测定以及MTT、LDH实验提示在西维来司钠三个剂量组之间存在明显的量-效关系,其对BMEC的保护作用有剂量依赖性。
结论:(1)我们成功的培养出了大鼠微血管内皮细胞,为建立离体血-脑屏障,研究脑血管疾病提供了帮助。
(2)建立了一种方便、稳定、可靠的体外细胞氧糖剥夺/复氧模型。
(3)明确了在脑缺血/再灌注损伤中,NE通过增加ET-1、ICAM-1、Tie-2等物质的表达,损伤脑微血管内皮细胞,进而造成脑微血管的损坏、血脑屏障的破坏,因而NE将成为脑血管疾病治疗一个新的作用靶点,这为NE特异抑制剂西维来司钠作为一种新型的神经保护剂用于脑血管疾病治疗的研究奠定了基础。
Background
Ischemic cerebrovascular disease is a kind of common and frequent disease that severely affects people’s lives and health. It is necessary to study the pathology and physiology mechanism deeply and search the pointed therapy strategy of ischemic cerebrovascular disease. It is important significance for economy and social.
Brain ischemia/reperfusion (I/R) has relationship with many factors, such as free radicals, the excitotoxicity, free calcium over-loading, the dysfunction of mitochondria, inflammation and apoptosis. Recent years, people gradually think highly of the roles of inflammation in brain ischemia/reperfusion injury. It is confirmed that the blood vessel inflammatory reaction by leucocyte (especially polymorphonuclear leukocyte ) and inflammatory cell factors make a important effect in I/R. The effect of polymorphonuclear leukocyte in I/R has two aspect:(1) mechanism blockage (2) releasing mediators of inflammation and proteolytic enzyme(neutrophil elastase) , aggraving the injury of brain tissue.
Neutrophil elastase(NE)is a kind of proteolytic enzyme which has intensive hydrolization and the most optimistic substrate is elastin. Previous study shows that neutrophil elastase either participates many inflammatory disease for example chronic bronchitis, bronchiectasis, gential tract infect or makes a important role in the I/R injury of tissue and organic.
At the same time, it is has proved that the damage of brain microvascular endothelial cells (BMEC) is the earlier pathological changes and basic remote cause in ischemic cerebrovascular disease. Therefore, we can further confirm that NE participate the ischemia /reperfusion injury,and the NE inhibitor Sivelestat Sodium likely to cerebral ischemia / reperfusion injury protection through the study of BMEC in oxygen glucose deprivation/reoxygenation.
Objective
(1) To isolate and to observe the morphology of brain microvascular endothelial cells from Wistar rat.
(2) To establish a simple, stable and reliable model of oxygen glucose deprivation/reoxygenation (OGD/R) of microvascular endothelial cells in vitro.
(3) To investigate whether NE aggtavate I/R injury though impairing brain blood capillary. So NE will become the new pointed therapy strategy in the therapy of ischemic cerebrovascular disease. And the Sivelestat sodium will be a new nerves protective additive in the therapy of cerebrovascular disease.
Methods
BMEC of 5～7 days Wistar rats were primary cultured. The BMEC were affirmed by immunocytochemical method, morphologies under light microscope and ultramicrostructure under electron microscope. We observed the growth curve of BMEC by MTT assay. We established a model of oxygen glucose deprivation/reoxygenation (OGD/R) of microvascular endothelial cells in vitro. The experiment has been divided 5 groups: normal control group (NC group); ischemia/reperfusion group (I/R group); ischemia/reperfusion + polymorphonuclear leukocyte group (I/R+PMN group); ischemia/reperfusion + polymorphonuclear leukocyte + Sivelestat sodium group (I/R+PMN+Siv group); polymorphonuclear leukocyte + Edaravone group (I/R+PMN+EDA group). We quantitative assayed the NE by ELASA; estimate cells survival and degree of injury by MTT and LDH; assayed the endothelin-1 secretory volume by radioactive immunoassay (RIA); detected the intercellular adhesion molecule-1 (ICAM-1) by Immuneeocytochemical method ,and assayed the angiogenin receptor-2 mRNA (Tie-2 mRNA) by RT-PCR.
Results
(1) The characterization of BMEC was confirmed based on the morphology and positive by the detection of factorⅧantigen.
(2) Oxygen glucose deprivation for 4 hours and reoxygenation for 24 hours can simulate the ischemia/reperfusion injury of BMEC in vivo.
(3)NE、MTT、LDH、ET-1、ICAM-1 and Tie-2experiment show: in the process of oxygen glucose deprivation/reoxygenation ,BMEC have subjected great damagement, I/R groups have significant differences compared with NC groups (p<0.05);polymorphonuclear leukocyte can aggregrate this injury, I/R+PMN groups have significant differences compared with I/R groups (p<0.05); after Sivelestat Sodium treatment, the effect of I/R injury has been decreased, I/R+PMN+Siv groups have significant differences compared with I/R+PMN groups (p<0.05), and NE、MTT、LDH experiment show that the three dose groups there was a significant dose-effect relationship. The protective effect on the BMEC has a dose-dependent manner.
Conlcusions
(1) We isolated and cultured BMEC in vitro successfully. The methods provided a useful way to study the cerebrovascular disease in vitro.
(2) We have established a simple, stable and reliable model of I/R of vascular endothelial cells in vitro successfully.
(3) It is confirmed that NE injury brain blood capillary and aggravate I/R brain injured through increasing the expression of ET-1、ICAM-1、Tie-2. So NE will become the new pointed therapy strategy in the therapy of ischemic cerebrovascular disease. And this will lay a foundation for the Sivelestat sodium as a new nerves protective additive in the therapy of cerebrovascular disease.
引文
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[5]Salvaterra CG,Goldman W F. Acute hypoxia increases cytosolic calcium in cultured pulmonary arterial myocytes [J]. Am J Physiol,1993, 264:L323-L328
[1]Mosmann T,Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytoxicity assay[J].Jimmunol Methods,1999,65:55-65
[2]Feuerstein G Z,W ang X. Inflammation and stroke:benefits without harm[J].Arch Neurol,2001,58(4):672-674
[3]Dinkel K,Dhabhar F S,Sapolsky R M .Neurotoxic efects of polymorphonuclear granulocytes on hippocampal primary cultures[J].Proc Natl Acad Sci USA,2004,101(1):331-336
[4]曾凡新 ,董志 ,傅洁民等. 中性粒细胞弹性蛋白酶—神经保护的作用新靶点[J]. 中国药理学通报,2006,22(7):784-787
[5]Zeiher BG,Matsuoka S,Kawabam K,et a1.Neutrophil elastase and acute lung inury : prospects for sivelestat and other neutrophil elastase inhibitors as therapeutics.Crit Care Med 2002;30(5 Supp1):S28l-287
[6]吴其标,曹世宏.中性粒细胞弹性蛋白酶在支气管扩张症发病中的作用及治疗策略[J].国外医学:内科学分册,2004,31(12):519-522
[7]Okajima K,Harada N,Uchiba M,et a1.Neutrophil elastase contributes to the development of ischemia-reperfusion-induced liver injury by decreasing endothelial production of prostacyclin in rats [J].Am J Physiol Gastrointest Liver Physiol,2004,287(6):1116- 23
[8]石晓星,商学军.中性粒细胞弹性蛋白酶在男性生殖道感染诊断中的意义[J].中华男科学,2003,9(2):136-139
[9]Chen H M,Chen J C,Shyr M H,et a1.Neutrophil elastase inhibitor(ONO-5046) attenuates reperfusion-induced hepatic microcirculatory derangement , energy depletion and lipid peroxidation in rats[J].Shock,1999,12(6):462-467
[10]Kotake Y,Yamamoto M ,Matsumoto M ,et a1. Sivelestat,a neutrophil elastase inhibitor , attenuates neutrophil priming after hepatoenteric ischemia in rabbits[J].Shock,2005,23(2):156-160
[11]Ginzberg H, Dong Q, Cantin A,et al. Neutrophil mediated epithelial injury during transmigration: role of elastase[J]. Am J Physiol,2001, 281:705-725
[12]Shapiro SD . Neutrophil elastase : path clearer , pathogen killer , or just pathologic[J].Am J Respir Cell Mol Biol,2002,26(3):266-268
[13]Yong RE,Thompson RD, Larbi KY, et al. Neutrophil elastase-deficient mice demonstrate a nonredundant role for NE in nuetrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo[J]. J Immunol, 2004, 172(7):4493-4502
[14] Tonai T,Shiba K,Taketani Y,et a1.A neutrophil elastase inhibitor (ONO-5046)reduces neurologic damage after spinal cord injury in rats[J].JNeurochem,2001,78(5):1064-1072
[15]Armao D,Komfeld M,Estrada E Y,et a1.Neutral proteases anddisruption of the blood-brain barrier in rat[J].Brain Res,1997, 767(2):259-64
[16]幺改琦,吴咸中.多形核粒细胞体外介导内皮细胞损伤的机制[J].中华实验外科杂志,1999,16(1):36-37
[17] 张予阳,刘岩,付守廷.脑缺血与炎症反应[J].中国药理学通报,2006。22(1):5-9
[18]袁海涛,田牛.油酸对培养血管内皮细胞的直接损伤作用.中国应用生理学杂志,1990,6(3):224-226
[19] Yang W ,Block E.Effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothdial cells.J Cell Physiol,1995,164:414-423
[20] Blann A D,Lip G Y.The endothelian in atherothrombetic diseases:assessment of function,mechanisms and clinical implications[J].Blood Coagul Fibrinodlysis,1998,9:297-306
[21]杜冠华.血管内皮细胞损伤机制及保护药物的研究[J].基础医学与临床,2004,24:258-261
[22]Basilico N,Speciale L,Parapini S,et a1.Endothelin-1 production by a microvascular endothelial cell line treated with plasmodium falciparum parasitized red blood cells[J].Clin Sci(Lond),2002,103(Suppl 48):s464-s466
[23]Kourembanas S,Marsden P A,Mcquillan L P,et a1.Hypoxia induced endothelin gene expression and secretion in cultured human endothelium [J].J Clin Invest,1991,88:1054-1060
[24]陈莉芬 ,陶 陶 ,余 震 等. 阿魏酸钠对缺氧诱导脑血管内皮细胞内皮素-1 表达的影响.中国中西医结合急救杂杂志,2005,12(6):344-346
[25] Steven D,Marlin A,Spring er W,et a1.Purified interceUular adhesion molecule- 1 is a ligand for lyphocyte function-associated antigen-1 [J].Cell,2000,51:813-819
[26] Matsuo Y,Onodra H,Shiga Y,et a1.Role of cell adhesion moecules in brain injury after transient cerebral artery occlusion in the rat brain[J].Res,1999,6:344-352
[27] Zhang RL,,Chopp M ,Jiang N,et a1.Anti-intercellular adhesion molecule-1 antibody reduces ischemic cell damage after transient but not permanent middle cerebral artery occlusion in the Wistar rat[J].Stroke,2000,26(8)1438-1443
[28]Yanagisawa M ,Kurihara H,Kimuras.et al.A novel potent vaso constrictor peptide procuced by vascular endothelial cues[J].Nature,1988;332:411
[29]Partanen J,Armstrong E,Makela TP,et a1.A novel endothelial surfaee receptor tyrosine kinase with extracellar epidermal growth factor homology domains[J]. Cell Biol,1992,12(4):1698-1707
[30]Sato TN,Tozawa Y,Deutsch U,et a1.Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation[J].Nature,1995.376 (6535):70-74
[31]Runting AS,Stacker SA,Wilks AF.Tie2,a putative protein tyrosine kinase from a new class of cell surface receptor[J].Growth Factors,1993,9(2):99-lO5
[32]Procopio WN,Pelavin PI,Lee MF,et a1.Angiopoietin-1 and-2 coiled-coil domains mediate distinct homo-oligomerization patterns,but fibrinogen like domains mediate ligand activity[J].J Biol Chem,1999,274(42): 30196-3020l
[33]Sato TN,Tozawa Y,Deutsch U,et a1.Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation[J].Nature,1995,376(6535):70-74
[34]Suri C,Jones PF,Patan S,et a1.Requisite role of angiopoietin-1,a ligand for the TIE-2 receptor,during embryonic angiogenesis[J].Cell,1996,87(7):1171-118O
[35]MORAES T J,CCOW C W,DOWNEY G P.Proteases and lung injury [J].Crit Care Med,2003,31(4 Supp1):S189-194
[36]Kaneko K,Kudoh I,Hattori S,et a1.Neutrophil elastase inhibitor,ONO-5046。modulates acid.induced lung and systemic injury in rabbits [J].Anesthesiology,1997,87(3):635-641
[37]Lee WL,Downey GP.Leukocyte elastase:physiological functions and role in acutelung injury[J].Am J Respir Crit Care Med,2001,164(5):896-904
[38]Kawabata K,Suzuki M,Sugitani M,et a1.ONO-5046,a novel inhibitor of human neutrophi1 elastase[J].Biochem Biophys Res Cornmun,1991,177(2):814-820
[39]张 红,苟大明,李健权等. 西维来司钠对体外循环中血浆弹性蛋白酶、补体C3、C4及尿NAG的影响[J].第三军医大学学报,2006,28(4):331-333
[1] Hess D,Howard E,Cheng C,et al.Hypertonic mannitol loading of NF--tcB transcription factor decoys in human brain microvascular endothelial cells blocks upregulation of ICAM-1[J].Stroke,2000,31(5):1179-1186
[2] Wang CX,Shuaib A.Involvement of inflammatory cytokines in central nervous system injury[J].Prog Neurobiol,2002;67(2):161
[3] Kostula N, Li HL,Xiao BG,et al. Dendritic cell are present in ischemic brain after permanent middle cerebral artery occlusion in the rats[J].Stroke,2002,33(4) ll29-35
[4] Montaner J,Molina C,Monasterio J,et al. Matrix metalloproteinase-9 pretreatment level predictsin- tracranial hemorrhagic complications after thrombolysis in human stroke[J].Circulation,2003,107: 598-603
[5] Chen L,Vicaut E,Sercombe RI. Polymorphonuclear leukocyte activation inducescerebral hypop- erfuslon in rats in the absence of previous isehemia-reperfusion damage[J]. Neurosci Lett ,2002, 33l(3):203-207
[6] 项洁,沈霞,耿德勤.肿瘤坏死因子-a 在大鼠脑缺血再灌注中的表达及对神经细胞凋亡的影响[J].中国临床康复,2004,8(28):6094-6095
[7] Zhang Jintao,Wu Weiping,Zhang Fengying,et a1.The Interleukin-1B Converting Enzyme Gene Expression after Cerebral Ischemia Reperfusion[J].Journal of Military Medicine,2002,27(1):56-58
[8] Bin Xiang, Liu Xiaowen, Liu Xinwen, et a1.Interleukir-1 Changing in Cerebral Cortex and Hippocampi after all Brain Ischemia RepeHusion[J].The Chinese Clinical Recovery,2002,6(1):50-51
[9] Auan SM,Parker LC,Co llins B,et a1.Cortical Cell Death Induced by IL- 1 is Mediated Via Actions in the Hypothalamus of the Rat[J].Proc Natl Acad Sci USA,2000,97(10):5580-5585
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[1]Thandroyen F T, Bellotto D,Katayama A,et a1. Subcellular electrolyte alterations during progressive hypoxia following reoxygenation in isolated neonatal rat ventricular myocytes [J]. Circ Res,1992,71(1):106-119
[2]Bredesen DE. Neural apoptosis[J]. Ann Neurol,1995,38(6):839-851
[3]李宏杰,章广玲,张连元等.一种用于研究骨骼肌缺血/再灌注损伤的细胞模型[J].Chin J Appl Physiol 2005,21(3):356-358
[4]Koyama T,Temma K,Akera T.Reperfusion-induced contracture develops with a decreasing [Ca2+] in single heart cells[J]. Am J Physiol,1991,261(4 pt 2):H l115-1122
[5]Salvaterra CG,Goldman W F. Acute hypoxia increases cytosolic calcium in cultured pulmonary arterial myocytes [J]. Am J Physiol,1993, 264:L323-L328
[1]Mosmann T,Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytoxicity assay[J].Jimmunol Methods,1999,65:55-65
[2]Feuerstein G Z,W ang X. Inflammation and stroke:benefits without harm[J].Arch Neurol,2001,58(4):672-674
[3]Dinkel K,Dhabhar F S,Sapolsky R M .Neurotoxic efects of polymorphonuclear granulocytes on hippocampal primary cultures[J].Proc Natl Acad Sci USA,2004,101(1):331-336
[4]曾凡新 ,董志 ,傅洁民等. 中性粒细胞弹性蛋白酶—神经保护的作用新靶点[J]. 中国药理学通报,2006,22(7):784-787
[5]Zeiher BG,Matsuoka S,Kawabam K,et a1.Neutrophil elastase and acute lung inury : prospects for sivelestat and other neutrophil elastase inhibitors as therapeutics.Crit Care Med 2002;30(5 Supp1):S28l-287
[6]吴其标,曹世宏.中性粒细胞弹性蛋白酶在支气管扩张症发病中的作用及治疗策略[J].国外医学:内科学分册,2004,31(12):519-522
[7]Okajima K,Harada N,Uchiba M,et a1.Neutrophil elastase contributes to the development of ischemia-reperfusion-induced liver injury by decreasing endothelial production of prostacyclin in rats [J].Am J Physiol Gastrointest Liver Physiol,2004,287(6):1116- 23
[8]石晓星,商学军.中性粒细胞弹性蛋白酶在男性生殖道感染诊断中的意义[J].中华男科学,2003,9(2):136-139
[9]Chen H M,Chen J C,Shyr M H,et a1.Neutrophil elastase inhibitor(ONO-5046) attenuates reperfusion-induced hepatic microcirculatory derangement , energy depletion and lipid peroxidation in rats[J].Shock,1999,12(6):462-467
[10]Kotake Y,Yamamoto M ,Matsumoto M ,et a1. Sivelestat,a neutrophil elastase inhibitor , attenuates neutrophil priming after hepatoenteric ischemia in rabbits[J].Shock,2005,23(2):156-160
[11]Ginzberg H, Dong Q, Cantin A,et al. Neutrophil mediated epithelial injury during transmigration: role of elastase[J]. Am J Physiol,2001, 281:705-725
[12]Shapiro SD . Neutrophil elastase : path clearer , pathogen killer , or just pathologic[J].Am J Respir Cell Mol Biol,2002,26(3):266-268
[13]Yong RE,Thompson RD, Larbi KY, et al. Neutrophil elastase-deficient mice demonstrate a nonredundant role for NE in nuetrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo[J]. J Immunol, 2004, 172(7):4493-4502
[14] Tonai T,Shiba K,Taketani Y,et a1.A neutrophil elastase inhibitor (ONO-5046)reduces neurologic damage after spinal cord injury in rats[J].JNeurochem,2001,78(5):1064-1072
[15]Armao D,Komfeld M,Estrada E Y,et a1.Neutral proteases anddisruption of the blood-brain barrier in rat[J].Brain Res,1997, 767(2):259-64
[16]幺改琦,吴咸中.多形核粒细胞体外介导内皮细胞损伤的机制[J].中华实验外科杂志,1999,16(1):36-37
[17] 张予阳,刘岩,付守廷.脑缺血与炎症反应[J].中国药理学通报,2006。22(1):5-9
[18]袁海涛,田牛.油酸对培养血管内皮细胞的直接损伤作用.中国应用生理学杂志,1990,6(3):224-226
[19] Yang W ,Block E.Effect of hypoxia and reoxygenation on the formation and release of reactive oxygen species by porcine pulmonary artery endothdial cells.J Cell Physiol,1995,164:414-423
[20] Blann A D,Lip G Y.The endothelian in atherothrombetic diseases:assessment of function,mechanisms and clinical implications[J].Blood Coagul Fibrinodlysis,1998,9:297-306
[21]杜冠华.血管内皮细胞损伤机制及保护药物的研究[J].基础医学与临床,2004,24:258-261
[22]Basilico N,Speciale L,Parapini S,et a1.Endothelin-1 production by a microvascular endothelial cell line treated with plasmodium falciparum parasitized red blood cells[J].Clin Sci(Lond),2002,103(Suppl 48):s464-s466
[23]Kourembanas S,Marsden P A,Mcquillan L P,et a1.Hypoxia induced endothelin gene expression and secretion in cultured human endothelium [J].J Clin Invest,1991,88:1054-1060
[24]陈莉芬 ,陶 陶 ,余 震 等. 阿魏酸钠对缺氧诱导脑血管内皮细胞内皮素-1 表达的影响.中国中西医结合急救杂杂志,2005,12(6):344-346
[25] Steven D,Marlin A,Spring er W,et a1.Purified interceUular adhesion molecule- 1 is a ligand for lyphocyte function-associated antigen-1 [J].Cell,2000,51:813-819
[26] Matsuo Y,Onodra H,Shiga Y,et a1.Role of cell adhesion moecules in brain injury after transient cerebral artery occlusion in the rat brain[J].Res,1999,6:344-352
[27] Zhang RL,,Chopp M ,Jiang N,et a1.Anti-intercellular adhesion molecule-1 antibody reduces ischemic cell damage after transient but not permanent middle cerebral artery occlusion in the Wistar rat[J].Stroke,2000,26(8)1438-1443
[28]Yanagisawa M ,Kurihara H,Kimuras.et al.A novel potent vaso constrictor peptide procuced by vascular endothelial cues[J].Nature,1988;332:411
[29]Partanen J,Armstrong E,Makela TP,et a1.A novel endothelial surfaee receptor tyrosine kinase with extracellar epidermal growth factor homology domains[J]. Cell Biol,1992,12(4):1698-1707
[30]Sato TN,Tozawa Y,Deutsch U,et a1.Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation[J].Nature,1995.376 (6535):70-74
[31]Runting AS,Stacker SA,Wilks AF.Tie2,a putative protein tyrosine kinase from a new class of cell surface receptor[J].Growth Factors,1993,9(2):99-lO5
[32]Procopio WN,Pelavin PI,Lee MF,et a1.Angiopoietin-1 and-2 coiled-coil domains mediate distinct homo-oligomerization patterns,but fibrinogen like domains mediate ligand activity[J].J Biol Chem,1999,274(42): 30196-3020l
[33]Sato TN,Tozawa Y,Deutsch U,et a1.Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation[J].Nature,1995,376(6535):70-74
[34]Suri C,Jones PF,Patan S,et a1.Requisite role of angiopoietin-1,a ligand for the TIE-2 receptor,during embryonic angiogenesis[J].Cell,1996,87(7):1171-118O
[35]MORAES T J,CCOW C W,DOWNEY G P.Proteases and lung injury [J].Crit Care Med,2003,31(4 Supp1):S189-194
[36]Kaneko K,Kudoh I,Hattori S,et a1.Neutrophil elastase inhibitor,ONO-5046。modulates acid.induced lung and systemic injury in rabbits [J].Anesthesiology,1997,87(3):635-641
[37]Lee WL,Downey GP.Leukocyte elastase:physiological functions and role in acutelung injury[J].Am J Respir Crit Care Med,2001,164(5):896-904
[38]Kawabata K,Suzuki M,Sugitani M,et a1.ONO-5046,a novel inhibitor of human neutrophi1 elastase[J].Biochem Biophys Res Cornmun,1991,177(2):814-820
[39]张 红,苟大明,李健权等. 西维来司钠对体外循环中血浆弹性蛋白酶、补体C3、C4及尿NAG的影响[J].第三军医大学学报,2006,28(4):331-333
[1] Hess D,Howard E,Cheng C,et al.Hypertonic mannitol loading of NF--tcB transcription factor decoys in human brain microvascular endothelial cells blocks upregulation of ICAM-1[J].Stroke,2000,31(5):1179-1186
[2] Wang CX,Shuaib A.Involvement of inflammatory cytokines in central nervous system injury[J].Prog Neurobiol,2002;67(2):161
[3] Kostula N, Li HL,Xiao BG,et al. Dendritic cell are present in ischemic brain after permanent middle cerebral artery occlusion in the rats[J].Stroke,2002,33(4) ll29-35
[4] Montaner J,Molina C,Monasterio J,et al. Matrix metalloproteinase-9 pretreatment level predictsin- tracranial hemorrhagic complications after thrombolysis in human stroke[J].Circulation,2003,107: 598-603
[5] Chen L,Vicaut E,Sercombe RI. Polymorphonuclear leukocyte activation inducescerebral hypop- erfuslon in rats in the absence of previous isehemia-reperfusion damage[J]. Neurosci Lett ,2002, 33l(3):203-207
[6] 项洁,沈霞,耿德勤.肿瘤坏死因子-a 在大鼠脑缺血再灌注中的表达及对神经细胞凋亡的影响[J].中国临床康复,2004,8(28):6094-6095
[7] Zhang Jintao,Wu Weiping,Zhang Fengying,et a1.The Interleukin-1B Converting Enzyme Gene Expression after Cerebral Ischemia Reperfusion[J].Journal of Military Medicine,2002,27(1):56-58
[8] Bin Xiang, Liu Xiaowen, Liu Xinwen, et a1.Interleukir-1 Changing in Cerebral Cortex and Hippocampi after all Brain Ischemia RepeHusion[J].The Chinese Clinical Recovery,2002,6(1):50-51
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