HIF-1α及VEGF在大鼠缺血缺氧性脑损伤中的表达研究
摘要
脑卒中(Cerebral Stroke)是最常见的神经系统致死性疾病,多年来,它以高发病率、高致残率和高病死率严重地威胁着人类的健康。其中缺血性脑卒中(Ischemic Cerebral Stroke)占绝大部分(约60-80%),目前临床上常见的溶栓及开颅手术等治疗受到诸多因素的限制仅对少数患者有效,因此进一步阐明其分子机制以及探索新的治疗方案已经迫在眉睫。
目的:本研究旨在建立了一种大鼠永久性大脑中动脉缺血模型。采用神经功能缺失行为学评分、TTC染色测定梗死范围、大鼠脑组织的组织病理学及电镜超微结构观察等对模型予以评价;并进一步探讨HIF-1α、VEGF在脑缺血缺氧性脑损伤中的表达规律及其分子机制,为探讨行之有效的治疗方法提供理论依据。
方法:
1动物分组:取体重250-280g健康成年SD大鼠80只,随机分3组:正常对照组大鼠5只,实验组50只,假手术组25只,按梗塞后不同取材时间分为6h、12h、24h、72h、7d 5个亚组。
2模型制备:实验组使用线栓法制备大鼠大脑中动脉缺血模型。假手术组除不插线栓外,操作步骤与实验组相同。各组大鼠术后行神经行为学评分,1～3分者入选实验。
3大鼠于预定时间点断头取脑,使用TTC染色观察脑梗塞范围。
4 HE染色光镜观察大脑中动脉缺血大鼠脑组织病理学改变,透射电镜观察神经元超微结构变化。
5免疫组化SP法测定各脑区HIF-1α、VEGF表达。显微镜下观察阳性表达情况,使用Image-Pro Plus 5.0图像分析系统测定每个视野阳性染色区域的平均光密度值。应用SPSS 15.0软件对结果进行统计学分析。
结果:
1成功制备了大鼠大脑中动脉缺血模型。栓塞后的大鼠均表现出不同程度的偏瘫症状,如身体倾斜、爬行旋转等症状。
2脑组织行TTC染色可见正常脑组织呈鲜红色,梗死区脑组织呈苍白色,并且随着梗塞时间的延长可见梗塞区扩大。
3大脑组织学及神经元超微结构的观察可见,24h后梗塞中心区出现大量神经元坏死,呈不可逆性,随着时间延长,坏死程度加重,坏死范围扩大;缺血半暗区部分神经元坏死,多数细胞结构受损但呈可修复性。
4 HIF-1α在正常对照组及假手术组偶可见少量阳性细胞。缺血6h,梗塞侧缺血中心区、缺血半暗区及梗塞对侧HIF-1α表达均增加;缺血12h,各脑区HIF-1α表达继续增高;缺血24h,梗塞侧皮层中心坏死区HIF-1α表达下降,而缺血半暗区及对侧皮层表达均继续增高并达高峰;缺血3d,各脑区HIF-1α的表达均开始下降。缺血7d,各脑区HIF-1α的表达继续降低到较低水平。统计学分析显示,HIF-1α蛋白水平的表达在正常对照组与假手术组之间无明显差异(P>0.05),而各实验组与正常对照组及假手术组比较显示HIF-1α表达水平明显增高,差异有统计学意义(P<0.05)。
5 VEGF在正常对照组和假手术组大鼠的脑组织中,VEGF仅见少量表达。缺血6h,梗塞侧缺血中心区、缺血半暗区及梗塞对侧VEGF蛋白水平表达均增加。缺血12h,梗塞侧皮层中心坏死区VEGF表达下降,而缺血半暗区及对侧皮层表达均继续增高;缺血24h,梗塞侧皮层中心坏死区VEGF表达继续降低,而缺血半暗区及梗塞对侧皮层VEGF表达继续增高并达高峰;缺血3d,各脑区VEGF的表达均下降;缺血7d,各脑区VEGF的表达持续下降至较低水平。统计学分析显示,VEGF蛋白水平的表达在正常对照组与假手术组之间无明显差异(P>0.05),而各实验组与正常对照组及假手术组之间比较显示VEGF表达水平明显增高,差异有统计学意义(P<0.05)。
6 HIF-1α与VEGF蛋白水平的表达在大鼠大脑中动脉缺血后的脑组织中呈正相关,相关系数r=0.7153,P<0.05,相关有统计学意义。
结论:
1使用线栓法可成功复制大脑中动脉缺血模型。经神经功能行为学评分、TTC染色、大体观察、HE染色组织学观察及电镜下细胞超微结构观察证实造模成功。
2脑梗塞24h-72h时间段,脑损伤程度最重,梗塞中心区神经元明显坏死,并且随着时间的延长,坏死程度渐加重,呈不可逆性;缺血半暗区部分神经元坏死,多数组织呈可修复性,尚存在挽救的机会。
3 HIF-1α只在一定氧浓度范围内表达,梗塞中心区完全缺氧情况下,神经元坏死后则无表达;缺血半暗区存在一定程度缺氧,HIF-1α在此处的神经元中存在明显高表达。
4 HIF-1α与VEGF于缺血梗塞24h时蛋白表达水平达到高峰,随梗塞时间的延长其表达水平呈峰样变化。
5在缺血梗塞后,HIF-1α与VEGF在脑组织中的表达水平随时间变化表现出升降一致的特点,二者的表达之间具有明显的相关性。
Cerebral Stroke is a very common fatal disease in nervous system. It threatens human’s health with its high attack rate, disability rate and case fatality rate. And ischemic Cerebral Stroke occupies about 60-80% of the total. Some therapies include thrombolysis and craniotomy are effective to few patients at present. Therefore, it’s imminent to illuminate the molecular mechanism and find a new therapeutic regimen.
Objective: This experimental study is for preparing a rat model with permanent middle cerebral artery occlusion (MCAO) and estimating the model by using the following methods as : ethology score of the loss of neural function, determining the infarction scope through TTC staining, observing the histopathology and ultramicrostructure of the rat’s brain tissues, and for finding the theory evidence of effective treatment by detecting the expression of HIF-1αand VEGF in the experimental ischemic brain tissues and observing the regularity of expression and molecular mechanism.
Methods:
1 Animals and groups: A total of 80 healthy adult Sprague-Dawley rats, weighting 250-280g, randomly divided into 3 groups: normal control group (n=5), experiment group (n=50), and sham operated group (n=25). The experiment group and the sham operated group divided into 5 subgroups by different infarction time: 6h, 12h, 24h, 72h, 7d.
2 Model preparation: Prepare the rat model with permanent middle cerebral artery occlusion (MCAO) using intraluminal suture by Zea Longa’s method. The operation step of sham operated group was same as experiment group except for plugging the suture in carotid artery. All rats were estimated by ethology score of the loss of neural function after operation. The animals whose score from 1-3 were selected to experiment group.
3 Animals in each group were killed at different preset time, and the brains were collected to observe the scope of cerebral infarction by TTC staining.
4 The brain tissues were collected to observe the histopathological change by HE Staining, to observe the changes of ultrastructure by electronic microscope.
5 The brain tissues were collected to detect the activation of HIF-1αand VEGF by immunohistochemistry. To observe the positive staining in every brain region by microscope, and to measure the MOD of positive area in every field of vision by“Image-Pro Plus 5.0”image analytical system. The results were calculated by statistical treatment with SPSS 15.0 software.
Results:
1 The rat model with middle cerebral artery occlusion (MCAO) was duplicated successfully. And the rats appeared hemiparalysis in different extent after infarction, such as body clinism, walk circuitation and so on.
2 The brain after TTC staining appeared red in normal tissue, and pale in infarction area. And the infarction zone was enlarged with the time extend.
3 It was found that lots of nerve cells in center of the infarction were necrosis obviously 24 hours after the infarction through observing the changes of histopathology structure and ultrastructure by electronic microscope. With the time extend, the degree of necrosis aggravated and the necrosis was inconvertibility. The nerve cells in the ischemic penumbra area were necrosis partly and could be repaired and saved.
4 The expression of HIF-1αon protein level: There were few positive cells in normal control and sham operated group. In ischemia 6h group: The expression of HIF-1αwas enhanced in center of infarction area, ischemic penumbra area and opposite side. In ischemia 12h group: the expression of HIF-1αwas enhanced continue. In ischemia 24h group: the expression of HIF-1αwas weakened in the center of infarction area. But the expression in ischemic penumbra area and opposite side were enhanced continue and to peak. In ischemia 3d group: the expression of HIF-1αin the three areas was all weakened. In ischemia 7d group: The expression of HIF-1αwere weakened continue and to a low level. There was no significantly difference in the expression of HIF-1αbetween normal control group and sham operated group (P>0.05). But the expression in the experiment group was higher significantly than the other two groups (P<0.05).
5 The expression of VEGF on protein level: There were A few positive cells in normal control and sham operated group. In ischemia 6h group: the expression of VEGF was enhanced in the center of infarction area, ischemic penumbra area and opposite side. In ischemia 12h group: the expression of VEGF was weakened in the center of infarction area. But the expression in ischemic penumbra area and opposite side were enhanced continue. In ischemia 24h group: the expression of VEGF was weakened continue in the center of infarction area. But the expression in ischemic penumbra area and opposite side were enhanced continue and to peak. In ischemia 3d group: the expression of VEGF in the three areas was all weakened. In ischemia 7d group: the expression of VEGF were weakened continue and to a low level. There was no significantly difference in the expression of VEGF between normal control group and sham operated group (P>0.05). But the expression in the experiment group was higher significantly than the other two groups (P<0.05).
6 The relation between HIF-1αand VEGF on protein level expression appears positive correlation. The coefficient correlation was 0.7153. There was statistical significance (P<0.05).
Conclusion:
1 The middle cerebral artery occlusion (MCAO) model was duplicated successfully using intraluminal suture and confirmed the success by neurology score, TTC staining, histological observation through HE staining and ultramicrostructure observation through electron microscope.
2 The nerve cells in the center of the infarction were necrosis obviously 24～72 hours after infarction,. As the time extended, the degree of necrosis aggravated and the necrosis was inconvertibility. The nerve cells in the ischemic penumbra area were necrosis partly and could be repaired and saved.
3 The expression of HIF-1αwas dependent on an oxygen concentration scope. In the center of the infarction, the nerve cells were hypoxia completely and there is no expression of HIF-1α. In the ischemic penumbra area, the hypoxia degree was not as the infarction center and the expression was obviously.
4 The expression peak of HIF-1αand VEGF appeared 24 hours after the infarction and the expression was changing as peak with the time extend.
5 After the infarction, the expression changing with time of HIF-1αwas coincidence with that of VEGF in brain tissues and the two factors were correlated. After the infarction, the expressing quantity of VEGF was higher than that of HIF-1αall the time.
目的:本研究旨在建立了一种大鼠永久性大脑中动脉缺血模型。采用神经功能缺失行为学评分、TTC染色测定梗死范围、大鼠脑组织的组织病理学及电镜超微结构观察等对模型予以评价;并进一步探讨HIF-1α、VEGF在脑缺血缺氧性脑损伤中的表达规律及其分子机制,为探讨行之有效的治疗方法提供理论依据。
方法:
1动物分组:取体重250-280g健康成年SD大鼠80只,随机分3组:正常对照组大鼠5只,实验组50只,假手术组25只,按梗塞后不同取材时间分为6h、12h、24h、72h、7d 5个亚组。
2模型制备:实验组使用线栓法制备大鼠大脑中动脉缺血模型。假手术组除不插线栓外,操作步骤与实验组相同。各组大鼠术后行神经行为学评分,1～3分者入选实验。
3大鼠于预定时间点断头取脑,使用TTC染色观察脑梗塞范围。
4 HE染色光镜观察大脑中动脉缺血大鼠脑组织病理学改变,透射电镜观察神经元超微结构变化。
5免疫组化SP法测定各脑区HIF-1α、VEGF表达。显微镜下观察阳性表达情况,使用Image-Pro Plus 5.0图像分析系统测定每个视野阳性染色区域的平均光密度值。应用SPSS 15.0软件对结果进行统计学分析。
结果:
1成功制备了大鼠大脑中动脉缺血模型。栓塞后的大鼠均表现出不同程度的偏瘫症状,如身体倾斜、爬行旋转等症状。
2脑组织行TTC染色可见正常脑组织呈鲜红色,梗死区脑组织呈苍白色,并且随着梗塞时间的延长可见梗塞区扩大。
3大脑组织学及神经元超微结构的观察可见,24h后梗塞中心区出现大量神经元坏死,呈不可逆性,随着时间延长,坏死程度加重,坏死范围扩大;缺血半暗区部分神经元坏死,多数细胞结构受损但呈可修复性。
4 HIF-1α在正常对照组及假手术组偶可见少量阳性细胞。缺血6h,梗塞侧缺血中心区、缺血半暗区及梗塞对侧HIF-1α表达均增加;缺血12h,各脑区HIF-1α表达继续增高;缺血24h,梗塞侧皮层中心坏死区HIF-1α表达下降,而缺血半暗区及对侧皮层表达均继续增高并达高峰;缺血3d,各脑区HIF-1α的表达均开始下降。缺血7d,各脑区HIF-1α的表达继续降低到较低水平。统计学分析显示,HIF-1α蛋白水平的表达在正常对照组与假手术组之间无明显差异(P>0.05),而各实验组与正常对照组及假手术组比较显示HIF-1α表达水平明显增高,差异有统计学意义(P<0.05)。
5 VEGF在正常对照组和假手术组大鼠的脑组织中,VEGF仅见少量表达。缺血6h,梗塞侧缺血中心区、缺血半暗区及梗塞对侧VEGF蛋白水平表达均增加。缺血12h,梗塞侧皮层中心坏死区VEGF表达下降,而缺血半暗区及对侧皮层表达均继续增高;缺血24h,梗塞侧皮层中心坏死区VEGF表达继续降低,而缺血半暗区及梗塞对侧皮层VEGF表达继续增高并达高峰;缺血3d,各脑区VEGF的表达均下降;缺血7d,各脑区VEGF的表达持续下降至较低水平。统计学分析显示,VEGF蛋白水平的表达在正常对照组与假手术组之间无明显差异(P>0.05),而各实验组与正常对照组及假手术组之间比较显示VEGF表达水平明显增高,差异有统计学意义(P<0.05)。
6 HIF-1α与VEGF蛋白水平的表达在大鼠大脑中动脉缺血后的脑组织中呈正相关,相关系数r=0.7153,P<0.05,相关有统计学意义。
结论:
1使用线栓法可成功复制大脑中动脉缺血模型。经神经功能行为学评分、TTC染色、大体观察、HE染色组织学观察及电镜下细胞超微结构观察证实造模成功。
2脑梗塞24h-72h时间段,脑损伤程度最重,梗塞中心区神经元明显坏死,并且随着时间的延长,坏死程度渐加重,呈不可逆性;缺血半暗区部分神经元坏死,多数组织呈可修复性,尚存在挽救的机会。
3 HIF-1α只在一定氧浓度范围内表达,梗塞中心区完全缺氧情况下,神经元坏死后则无表达;缺血半暗区存在一定程度缺氧,HIF-1α在此处的神经元中存在明显高表达。
4 HIF-1α与VEGF于缺血梗塞24h时蛋白表达水平达到高峰,随梗塞时间的延长其表达水平呈峰样变化。
5在缺血梗塞后,HIF-1α与VEGF在脑组织中的表达水平随时间变化表现出升降一致的特点,二者的表达之间具有明显的相关性。
Cerebral Stroke is a very common fatal disease in nervous system. It threatens human’s health with its high attack rate, disability rate and case fatality rate. And ischemic Cerebral Stroke occupies about 60-80% of the total. Some therapies include thrombolysis and craniotomy are effective to few patients at present. Therefore, it’s imminent to illuminate the molecular mechanism and find a new therapeutic regimen.
Objective: This experimental study is for preparing a rat model with permanent middle cerebral artery occlusion (MCAO) and estimating the model by using the following methods as : ethology score of the loss of neural function, determining the infarction scope through TTC staining, observing the histopathology and ultramicrostructure of the rat’s brain tissues, and for finding the theory evidence of effective treatment by detecting the expression of HIF-1αand VEGF in the experimental ischemic brain tissues and observing the regularity of expression and molecular mechanism.
Methods:
1 Animals and groups: A total of 80 healthy adult Sprague-Dawley rats, weighting 250-280g, randomly divided into 3 groups: normal control group (n=5), experiment group (n=50), and sham operated group (n=25). The experiment group and the sham operated group divided into 5 subgroups by different infarction time: 6h, 12h, 24h, 72h, 7d.
2 Model preparation: Prepare the rat model with permanent middle cerebral artery occlusion (MCAO) using intraluminal suture by Zea Longa’s method. The operation step of sham operated group was same as experiment group except for plugging the suture in carotid artery. All rats were estimated by ethology score of the loss of neural function after operation. The animals whose score from 1-3 were selected to experiment group.
3 Animals in each group were killed at different preset time, and the brains were collected to observe the scope of cerebral infarction by TTC staining.
4 The brain tissues were collected to observe the histopathological change by HE Staining, to observe the changes of ultrastructure by electronic microscope.
5 The brain tissues were collected to detect the activation of HIF-1αand VEGF by immunohistochemistry. To observe the positive staining in every brain region by microscope, and to measure the MOD of positive area in every field of vision by“Image-Pro Plus 5.0”image analytical system. The results were calculated by statistical treatment with SPSS 15.0 software.
Results:
1 The rat model with middle cerebral artery occlusion (MCAO) was duplicated successfully. And the rats appeared hemiparalysis in different extent after infarction, such as body clinism, walk circuitation and so on.
2 The brain after TTC staining appeared red in normal tissue, and pale in infarction area. And the infarction zone was enlarged with the time extend.
3 It was found that lots of nerve cells in center of the infarction were necrosis obviously 24 hours after the infarction through observing the changes of histopathology structure and ultrastructure by electronic microscope. With the time extend, the degree of necrosis aggravated and the necrosis was inconvertibility. The nerve cells in the ischemic penumbra area were necrosis partly and could be repaired and saved.
4 The expression of HIF-1αon protein level: There were few positive cells in normal control and sham operated group. In ischemia 6h group: The expression of HIF-1αwas enhanced in center of infarction area, ischemic penumbra area and opposite side. In ischemia 12h group: the expression of HIF-1αwas enhanced continue. In ischemia 24h group: the expression of HIF-1αwas weakened in the center of infarction area. But the expression in ischemic penumbra area and opposite side were enhanced continue and to peak. In ischemia 3d group: the expression of HIF-1αin the three areas was all weakened. In ischemia 7d group: The expression of HIF-1αwere weakened continue and to a low level. There was no significantly difference in the expression of HIF-1αbetween normal control group and sham operated group (P>0.05). But the expression in the experiment group was higher significantly than the other two groups (P<0.05).
5 The expression of VEGF on protein level: There were A few positive cells in normal control and sham operated group. In ischemia 6h group: the expression of VEGF was enhanced in the center of infarction area, ischemic penumbra area and opposite side. In ischemia 12h group: the expression of VEGF was weakened in the center of infarction area. But the expression in ischemic penumbra area and opposite side were enhanced continue. In ischemia 24h group: the expression of VEGF was weakened continue in the center of infarction area. But the expression in ischemic penumbra area and opposite side were enhanced continue and to peak. In ischemia 3d group: the expression of VEGF in the three areas was all weakened. In ischemia 7d group: the expression of VEGF were weakened continue and to a low level. There was no significantly difference in the expression of VEGF between normal control group and sham operated group (P>0.05). But the expression in the experiment group was higher significantly than the other two groups (P<0.05).
6 The relation between HIF-1αand VEGF on protein level expression appears positive correlation. The coefficient correlation was 0.7153. There was statistical significance (P<0.05).
Conclusion:
1 The middle cerebral artery occlusion (MCAO) model was duplicated successfully using intraluminal suture and confirmed the success by neurology score, TTC staining, histological observation through HE staining and ultramicrostructure observation through electron microscope.
2 The nerve cells in the center of the infarction were necrosis obviously 24～72 hours after infarction,. As the time extended, the degree of necrosis aggravated and the necrosis was inconvertibility. The nerve cells in the ischemic penumbra area were necrosis partly and could be repaired and saved.
3 The expression of HIF-1αwas dependent on an oxygen concentration scope. In the center of the infarction, the nerve cells were hypoxia completely and there is no expression of HIF-1α. In the ischemic penumbra area, the hypoxia degree was not as the infarction center and the expression was obviously.
4 The expression peak of HIF-1αand VEGF appeared 24 hours after the infarction and the expression was changing as peak with the time extend.
5 After the infarction, the expression changing with time of HIF-1αwas coincidence with that of VEGF in brain tissues and the two factors were correlated. After the infarction, the expressing quantity of VEGF was higher than that of HIF-1αall the time.
引文
1 Semenza GL. Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology (Bethesda), 2004, 19:176-182
2 Brahimi-Horn, J. Pouyssegur, Harnessing the hypoxia-inducible factor in cancer and ischemic disease. Biochem Pharmacol. 2007, 73(3): 450-457
3 Papandreou I, Powell A, Lim AL, et al. Cellular reaction to hypoxia: sensing and responding to an adverse environment. Mutat Res, 2005, 569(1-2):87-100
4 Storkebaum E, Lambrechts D, Carmeliet P. VEGF: once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays. 2004 ,26(9):943-954
5 Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989, 20(1):84-91
6 Koizumi J, Yoshida Y, Nakazawa T, et al. Experimental studies of ischemic brain edema, I: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn J Stroke,, 8(1):1-8
7 Nagasawa H, Kogure K. Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion. Stroke, 1989, 20(8):1037-1043
8 Kuge Y, Minematsu K, Yamaguchi T, et al. Nylonmonofilament for intraluminal middle cerebral artery occlusion in rats. Stroke, 1995, 26(9):1655-1657
9 Wang LC, Futrell N, Wang DZ, et al. A reproducible model of middle cerebral infarcts, compatible with long-term survival, in aged rats. Stroke, 1995, 26(11):2087-2090
10 Garcia JH, Liu KF, Yoshida Y, et al. Brain microvessels: factors altering their patency after the occlusion of a middle cerebral artery (Wistar rat). Am J Pathol, 1994, 145(3):728-740
11 Kitagawa K, Matsumoto M, Tagaya M, et al. Ischemic tolerance' phenomenon found in the brain. Brain Res, 1990, 528(1):21-24
12 Barbe MF, Tytell M, Gower DJ, et al. Hyperthermia protects against light damage in the rat retina. Science, 1988, 241(4874):1817-1820
13 Bederson JB, Pitts LH, Germano SM, et al. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke, 1986, 17(6):1304-1308
14 Marti HJ, Bernaudin M, Bellail A, et al. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am J Pathol, 2000, 156(3):965-976
15 Semenza GL, Roth PH, Fang HM, et al. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem, 1994,269(38):23757-23763
16 Papandreou I, Powell A, Lim AL, et al. Cellular reaction to hypoxia: sensing and responding to an adverse environment. Mutat Res, 2005, 569(1-2):87-100
17 Chandel NS, Budinger GR. The cellular basis for diverse responses to oxygen. Free Radic Biol Med, 2007, 42(2):165-174
18 Lee JW, Bae SH, Jeong JW, et al. Hypoxia-inducible factor (HIF-1) alpha: its protein stability and biological functions. Exp Mol Med, 2004, 36(1):1-12
19 Semenza GL. O2-regulated gene expression: transcriptional control of cardiorespiratory physiology by HIF-1. J Appl Physiol, 2004, 96(3):1173-1177
20 Semenza GL. Oxygen-regulated transcription factors and their role in pulmonary disease. Respir Res, 2000, 1(3):159-162
21 Stroka DM, Burkhardt T, Desbaillets I, et al. HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia. FASEB J, 2001, 15(3):2445-2453
22 Ratan RR, Siddiq A, Smirnova N, et al. Harnessing hypoxic adaptation to prevent, treat, and repair stroke. J Mol Med. 2007,85(12):1331-1338
23 Bergeron M, Yu AY, Solway KE, et al. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur J Neurosci, 1999,11(12):4159-4170
24 Metzen E, Berchner-Pfannschmidt U, Stengel P,et al. Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing. J Cell Sci, 2003, 116(Pt 7):1319-1326
25 Gendron A, Kouassi E, Nuara S, et al. Transient middle cerebral artery occlusion influence on systemic oxygen homeostasis and erythropoiesis in Wistar rats. Stroke, 2004, 35:1979-1984
26 Sharp FR, Lu A, Tang Y, et al. Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab, 2000, 20(7):1011-1032
27 Mu D, Jiang X, Sheldon RA, et al. Regulation of hypoxia-inducible factor 1alpha and induction of vascular endothelial growth factor in a rat neonatal stroke model. Neurobiol Dis, 2003, 14(3):524-534
28 Matsuda T, Abe T, Wu JL, et al. Hypoxia-inducible factor-1alpha DNA induced angiogenesis in a rat cerebral ischemia model. Neurol Res, 2005, 27(5):503-508
1 Semenza GL. Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology (Bethesda), 2004, 19:176-182
2 Brahimi-Horn, J. Pouyssegur, Harnessing the hypoxia-inducible factor in cancer and ischemic disease. Biochem Pharmacol. 2007, 73(3): 450-457
3 Semenza GL, Roth PH, Fang HM, et al. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem, 1994, 269(38): 23757-23763
4 Lee JW, Bae SH, Jeong JW, et al. Hypoxia-inducible factor (HIF-1) alpha: its protein stability and biological functions. Exp Mol Med, 2004, 36(1):1-12
5 Papandreou I, Powell A, Lim AL, et al. Cellular reaction to hypoxia: sensing and responding to an adverse environment. Mutat Res, 2005, 569(1-2):87-100
6 Chandel NS, Budinger GR. The cellular basis for diverse responses to oxygen. Free Radic Biol Med, 2007, 42(2):165-74
7 Ehrismann D, Flashman E, Genn DN, et al. Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay. Biochem J, 2007, 401(1):227–234
8 Semenza GL. O2-regulated gene expression: transcriptionalcontrol of cardiorespiratory physiology by HIF-1. J Appl Physiol, 2004, 96(3):1173-1177
9 Semenza GL. Oxygen-regulated transcription factors and their role in pulmonary disease. Respir Res, 2000, 1(3):159-162
10 Stroka DM, Burkhardt T, Desbaillets I, et al. HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia. FASEB J, 2001, 15(3):2445-2453
11 Vengellur A, LaPres JJ. The role of hypoxia inducible factor 1alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts. Toxicol Sci, 2004, 82(2):638-646
12 McNeill LA, Hewitson KS, Gleadle JM, et al. The use of dioxygen by HIF prolyl hydroxylase (PHD1). Bioorg Med Chem Lett, 2002, 12(12):1547-1550
13 Metzen E, Zhou J, Jelkmann W, et al. Nitric oxide impairs normoxic degradation of HIF-1alpha by inhibition of prolyl hydroxylases. Mol Biol Cell, 2003, 14(8):3470-3481
14 Mazure NM, Brahimi-Horn MC, Pouyssegur J. Protein kinases and the hypoxia-inducible factor-1, two switches in angiogenesis. Curr Pharm Des, 2003, 9:531-541
15 Manalo DJ, Rowan A, Lavoie T, et al. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood, 2005,105(2):659-669
16 Pawlak S, Pawlak D, Buczko W. Hypoxia involvement in erythropoiesis regulation--a new insight. Przegl Lek, 2004,61(12):1415-1419
17 Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer, 2003, 3(10):721-732
18 Goda N, Ryan HE, Khadivi B, et al. Hypoxia-inducible factor 1alpha is essential for cell cycle arrest during hypoxia. Mol Cell Biol, 2003, 23(1):359-369
19 Greijer AE, van der Wall E. The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. J Clin Pathol, 2004, 57(10):1009-1014
20 Metzen E, Berchner-Pfannschmidt U, Stengel P,et al. Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing. J Cell Sci, 2003, 116(Pt 7):1319-1326
21 Sharp FR, Lu A, Tang Y, et al. Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab, 2000, 20(7):1011-1032
22 Bergeron M, Yu AY, Solway KE, et al. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur J Neurosci, 1999, 11(12):4159-4170
23 Sharp FR, Bergeron M, Bernaudin M. Hypoxia-inducible factor in brain. Adv Exp Med Biol, 2001, 502:273-291
24 Bernaudin M, Nedelec AS, Divoux D, et al. Normobaric hypoxia induces tolerance to focal permanent cerebral ischemia in association with an increased expression of hypoxia-inducible factor-1 and its target genes,erythropoietin and VEGF, in the adult mouse brain. J Cereb Blood Flow Metab, 2002, 22(4):393-403
25 Prass K, Scharff A, Ruscher K, et al. Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin. Stroke, 2003, 34(8):1981-1986
26 Liu Q, Berchner-Pfannschmidt U, Moller U, et al. A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression. Proc Natl Acad Sci U S A, 2004, 101(12):4302-4307
27 Ratan RR, Siddiq A, Smirnova N, et al. Harnessing hypoxic adaptation to prevent, treat, and repair stroke. J Mol Med. 2007 Dec;85(12):1331-1338
28 Mu D, Jiang X, Sheldon RA, et al. Regulation of hypoxia-inducible factor 1alpha and induction of vascular endothelial growth factor in a rat neonatal stroke model. Neurobiol Dis, 2003, 14(3):524-534
29 Chong ZZ, Kang JQ, Maiese K. Erythropoietin is a novel vascular protectant through activation of Akt1 and mitochondrial modulation of cysteine proteases. Circulation, 2002, 106(23):2973-2979
30 Matsuda T, Abe T, Wu JL, et al. Hypoxia-inducible factor-1alpha DNA induced angiogenesis in a rat cerebral ischemia model. Neurol Res, 2005, 27(5):503-508
31 Nangaku M, Izuhara Y, Takizawa S, et, al. A novel class of prolyl hydroxylase inhibitors induces angiogenesis and exerts organ protection against ischemia. ArteriosclerThromb Vasc Biol. 2007 Dec;27(12):2548-2554
32 Li L, Qu Y, Li J, et, al. Relationship between HIF-1alpha expression and neuronal apoptosis in neonatal rats with hypoxia-ischemia brain injury. Brain Res. 2007 ,14(1180):133-139
33 Heinl-Green A, Radke PW, Munkonge FM, et al. The efficacy of a 'master switch gene' HIF-1alpha in a porcine model of chronic myocardial ischaemia. Eur Heart J, 2005, 26(13):1327-1332
34 Patel TH, Kimura H,Weiss CR, et al. Constitutively active HIF-1α improves perfusion and arterial remodeling in an endovascular model of limb ischemia. Cardiovasc Res, 2005,68(1): 144–154
35 Rajagopalan S, Olin J, Deitcher S, et al. Use of a constitutively active hypoxia-inducible factor-1alpha transgene as a therapeutic strategy in no-option critical limb ischemia patients: phase I dose-escalation experience. Circulation , 2007, 115(10), 1234–1243
36 Semenza GL. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends Mol Med, 2001, 7(8):345-350
37 谭新杰,胡长林.缺氧诱导因子-1α 基因在成年大鼠局灶性脑 缺 血 中 的 治 疗 作 用 研 究 . 中 华 医 学 杂 志 , 2006, 15(86):1057-60
2 Brahimi-Horn, J. Pouyssegur, Harnessing the hypoxia-inducible factor in cancer and ischemic disease. Biochem Pharmacol. 2007, 73(3): 450-457
3 Papandreou I, Powell A, Lim AL, et al. Cellular reaction to hypoxia: sensing and responding to an adverse environment. Mutat Res, 2005, 569(1-2):87-100
4 Storkebaum E, Lambrechts D, Carmeliet P. VEGF: once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays. 2004 ,26(9):943-954
5 Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989, 20(1):84-91
6 Koizumi J, Yoshida Y, Nakazawa T, et al. Experimental studies of ischemic brain edema, I: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn J Stroke,, 8(1):1-8
7 Nagasawa H, Kogure K. Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion. Stroke, 1989, 20(8):1037-1043
8 Kuge Y, Minematsu K, Yamaguchi T, et al. Nylonmonofilament for intraluminal middle cerebral artery occlusion in rats. Stroke, 1995, 26(9):1655-1657
9 Wang LC, Futrell N, Wang DZ, et al. A reproducible model of middle cerebral infarcts, compatible with long-term survival, in aged rats. Stroke, 1995, 26(11):2087-2090
10 Garcia JH, Liu KF, Yoshida Y, et al. Brain microvessels: factors altering their patency after the occlusion of a middle cerebral artery (Wistar rat). Am J Pathol, 1994, 145(3):728-740
11 Kitagawa K, Matsumoto M, Tagaya M, et al. Ischemic tolerance' phenomenon found in the brain. Brain Res, 1990, 528(1):21-24
12 Barbe MF, Tytell M, Gower DJ, et al. Hyperthermia protects against light damage in the rat retina. Science, 1988, 241(4874):1817-1820
13 Bederson JB, Pitts LH, Germano SM, et al. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke, 1986, 17(6):1304-1308
14 Marti HJ, Bernaudin M, Bellail A, et al. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am J Pathol, 2000, 156(3):965-976
15 Semenza GL, Roth PH, Fang HM, et al. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem, 1994,269(38):23757-23763
16 Papandreou I, Powell A, Lim AL, et al. Cellular reaction to hypoxia: sensing and responding to an adverse environment. Mutat Res, 2005, 569(1-2):87-100
17 Chandel NS, Budinger GR. The cellular basis for diverse responses to oxygen. Free Radic Biol Med, 2007, 42(2):165-174
18 Lee JW, Bae SH, Jeong JW, et al. Hypoxia-inducible factor (HIF-1) alpha: its protein stability and biological functions. Exp Mol Med, 2004, 36(1):1-12
19 Semenza GL. O2-regulated gene expression: transcriptional control of cardiorespiratory physiology by HIF-1. J Appl Physiol, 2004, 96(3):1173-1177
20 Semenza GL. Oxygen-regulated transcription factors and their role in pulmonary disease. Respir Res, 2000, 1(3):159-162
21 Stroka DM, Burkhardt T, Desbaillets I, et al. HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia. FASEB J, 2001, 15(3):2445-2453
22 Ratan RR, Siddiq A, Smirnova N, et al. Harnessing hypoxic adaptation to prevent, treat, and repair stroke. J Mol Med. 2007,85(12):1331-1338
23 Bergeron M, Yu AY, Solway KE, et al. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur J Neurosci, 1999,11(12):4159-4170
24 Metzen E, Berchner-Pfannschmidt U, Stengel P,et al. Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing. J Cell Sci, 2003, 116(Pt 7):1319-1326
25 Gendron A, Kouassi E, Nuara S, et al. Transient middle cerebral artery occlusion influence on systemic oxygen homeostasis and erythropoiesis in Wistar rats. Stroke, 2004, 35:1979-1984
26 Sharp FR, Lu A, Tang Y, et al. Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab, 2000, 20(7):1011-1032
27 Mu D, Jiang X, Sheldon RA, et al. Regulation of hypoxia-inducible factor 1alpha and induction of vascular endothelial growth factor in a rat neonatal stroke model. Neurobiol Dis, 2003, 14(3):524-534
28 Matsuda T, Abe T, Wu JL, et al. Hypoxia-inducible factor-1alpha DNA induced angiogenesis in a rat cerebral ischemia model. Neurol Res, 2005, 27(5):503-508
1 Semenza GL. Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology (Bethesda), 2004, 19:176-182
2 Brahimi-Horn, J. Pouyssegur, Harnessing the hypoxia-inducible factor in cancer and ischemic disease. Biochem Pharmacol. 2007, 73(3): 450-457
3 Semenza GL, Roth PH, Fang HM, et al. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem, 1994, 269(38): 23757-23763
4 Lee JW, Bae SH, Jeong JW, et al. Hypoxia-inducible factor (HIF-1) alpha: its protein stability and biological functions. Exp Mol Med, 2004, 36(1):1-12
5 Papandreou I, Powell A, Lim AL, et al. Cellular reaction to hypoxia: sensing and responding to an adverse environment. Mutat Res, 2005, 569(1-2):87-100
6 Chandel NS, Budinger GR. The cellular basis for diverse responses to oxygen. Free Radic Biol Med, 2007, 42(2):165-74
7 Ehrismann D, Flashman E, Genn DN, et al. Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay. Biochem J, 2007, 401(1):227–234
8 Semenza GL. O2-regulated gene expression: transcriptionalcontrol of cardiorespiratory physiology by HIF-1. J Appl Physiol, 2004, 96(3):1173-1177
9 Semenza GL. Oxygen-regulated transcription factors and their role in pulmonary disease. Respir Res, 2000, 1(3):159-162
10 Stroka DM, Burkhardt T, Desbaillets I, et al. HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia. FASEB J, 2001, 15(3):2445-2453
11 Vengellur A, LaPres JJ. The role of hypoxia inducible factor 1alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts. Toxicol Sci, 2004, 82(2):638-646
12 McNeill LA, Hewitson KS, Gleadle JM, et al. The use of dioxygen by HIF prolyl hydroxylase (PHD1). Bioorg Med Chem Lett, 2002, 12(12):1547-1550
13 Metzen E, Zhou J, Jelkmann W, et al. Nitric oxide impairs normoxic degradation of HIF-1alpha by inhibition of prolyl hydroxylases. Mol Biol Cell, 2003, 14(8):3470-3481
14 Mazure NM, Brahimi-Horn MC, Pouyssegur J. Protein kinases and the hypoxia-inducible factor-1, two switches in angiogenesis. Curr Pharm Des, 2003, 9:531-541
15 Manalo DJ, Rowan A, Lavoie T, et al. Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood, 2005,105(2):659-669
16 Pawlak S, Pawlak D, Buczko W. Hypoxia involvement in erythropoiesis regulation--a new insight. Przegl Lek, 2004,61(12):1415-1419
17 Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer, 2003, 3(10):721-732
18 Goda N, Ryan HE, Khadivi B, et al. Hypoxia-inducible factor 1alpha is essential for cell cycle arrest during hypoxia. Mol Cell Biol, 2003, 23(1):359-369
19 Greijer AE, van der Wall E. The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. J Clin Pathol, 2004, 57(10):1009-1014
20 Metzen E, Berchner-Pfannschmidt U, Stengel P,et al. Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing. J Cell Sci, 2003, 116(Pt 7):1319-1326
21 Sharp FR, Lu A, Tang Y, et al. Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab, 2000, 20(7):1011-1032
22 Bergeron M, Yu AY, Solway KE, et al. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur J Neurosci, 1999, 11(12):4159-4170
23 Sharp FR, Bergeron M, Bernaudin M. Hypoxia-inducible factor in brain. Adv Exp Med Biol, 2001, 502:273-291
24 Bernaudin M, Nedelec AS, Divoux D, et al. Normobaric hypoxia induces tolerance to focal permanent cerebral ischemia in association with an increased expression of hypoxia-inducible factor-1 and its target genes,erythropoietin and VEGF, in the adult mouse brain. J Cereb Blood Flow Metab, 2002, 22(4):393-403
25 Prass K, Scharff A, Ruscher K, et al. Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin. Stroke, 2003, 34(8):1981-1986
26 Liu Q, Berchner-Pfannschmidt U, Moller U, et al. A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression. Proc Natl Acad Sci U S A, 2004, 101(12):4302-4307
27 Ratan RR, Siddiq A, Smirnova N, et al. Harnessing hypoxic adaptation to prevent, treat, and repair stroke. J Mol Med. 2007 Dec;85(12):1331-1338
28 Mu D, Jiang X, Sheldon RA, et al. Regulation of hypoxia-inducible factor 1alpha and induction of vascular endothelial growth factor in a rat neonatal stroke model. Neurobiol Dis, 2003, 14(3):524-534
29 Chong ZZ, Kang JQ, Maiese K. Erythropoietin is a novel vascular protectant through activation of Akt1 and mitochondrial modulation of cysteine proteases. Circulation, 2002, 106(23):2973-2979
30 Matsuda T, Abe T, Wu JL, et al. Hypoxia-inducible factor-1alpha DNA induced angiogenesis in a rat cerebral ischemia model. Neurol Res, 2005, 27(5):503-508
31 Nangaku M, Izuhara Y, Takizawa S, et, al. A novel class of prolyl hydroxylase inhibitors induces angiogenesis and exerts organ protection against ischemia. ArteriosclerThromb Vasc Biol. 2007 Dec;27(12):2548-2554
32 Li L, Qu Y, Li J, et, al. Relationship between HIF-1alpha expression and neuronal apoptosis in neonatal rats with hypoxia-ischemia brain injury. Brain Res. 2007 ,14(1180):133-139
33 Heinl-Green A, Radke PW, Munkonge FM, et al. The efficacy of a 'master switch gene' HIF-1alpha in a porcine model of chronic myocardial ischaemia. Eur Heart J, 2005, 26(13):1327-1332
34 Patel TH, Kimura H,Weiss CR, et al. Constitutively active HIF-1α improves perfusion and arterial remodeling in an endovascular model of limb ischemia. Cardiovasc Res, 2005,68(1): 144–154
35 Rajagopalan S, Olin J, Deitcher S, et al. Use of a constitutively active hypoxia-inducible factor-1alpha transgene as a therapeutic strategy in no-option critical limb ischemia patients: phase I dose-escalation experience. Circulation , 2007, 115(10), 1234–1243
36 Semenza GL. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends Mol Med, 2001, 7(8):345-350
37 谭新杰,胡长林.缺氧诱导因子-1α 基因在成年大鼠局灶性脑 缺 血 中 的 治 疗 作 用 研 究 . 中 华 医 学 杂 志 , 2006, 15(86):1057-60