实验性脑内血肿吸收机制及其干预研究
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摘要
目的
目前认为治疗脑出血(intracerebral hemorrhage,ICH)的根本措施就是促进脑内血肿的尽早吸收、控制脑水肿和减轻或逆转血肿周围组织的损伤。但针对血肿本身的治疗研究不多，临床上除手术清除血肿外，内科治疗尚缺乏促进血肿迅速溶解吸收的有效药物，基本上属对症治疗。因此，寻找既能促进脑内血肿迅速溶解，又能促进胶质增生，从而加快坏死组织的清除、分泌多种神经营养因子对血肿周围神经组织损伤有干预作用的药物，来延长受损神经元的存活和加快自身修复，是ICH急性期非手术治疗脑内血肿、保护因缺血引起的神经元损伤，降低死亡率、减少致残率的关键。已有人用水蛭或经炮制过的全水蛭粗提品对ICH或脑梗死进行过实验和临床研究，发现它有清除自由基、降低血浆内皮素含量、增加血浆降钙素基因相关肽含量、抗凋亡、改善脑血流量等功效。目前对缺血性心脑血管病的治疗已成共识，也有少数全水蛭中药复合物治疗ICH的报道，提示它有缩短血肿吸收时间的作用，亦未发现该药引起凝血机制正常者的再出血副作用。但他们都未从水蛭素是凝血酶最强的特异性抑制剂这一最重要的药理性质出发，考察水蛭提取液对凝血、纤溶平衡的调节来探索其消除血肿，加快自身修复的可能机制。因此，我们的研究目的就是结合体内与体外实验，从整体、组织、细胞到分子生物学水平来研究脑内血肿自然吸收的规律与机制，试图证实水蛭提取液对急性期实验性脑内血肿的吸收有促进作用，并揭示其可能的作用机制，观察其毒副作用，为其临床治疗脑内血肿提供更多的实验依据。方法：本研究拟采用体内实验即胶原酶诱导大鼠ICH模型，体外实验即大鼠大脑皮质BMEC和As培养为实验模型，选多项观察指标包括实验鼠神经功能缺损体征评分；病理改变(普通光镜、电镜与脑微血管造影)；脑水含量、血肿容积与血肿周围缺血体积测量；生化指标(tPA、PAI-1、D-D、Fbg、PT、aPTT)检测；免疫组化及RT-PCR检测。结果：(1) 大鼠 ICH后急性期血肿周围存在缺血性损害。(2) 脑内血肿随时间推移逐渐缩小，但需10d方能明显缩小，14d时仍有部分大鼠血肿尚未完全
吸收。ICH后第3d、第6d血肿周围脑组织tPA含量、活性与mRNA表达增加及aPTT延长，且呈时间依赖性。PAI-1含量与活性或mRNA表达无明显抑制。(3) 水蛭提取液能促进脑内血肿的吸收，明显吸收发生于治疗后第6d，同时能缩小血肿周围缺血范围、降低脑水含量。对ICH后神经功能的恢复有明显的促进作用。病理检查发现它能促进病灶周围血管内皮细胞、毛细血管和胶质细胞增生，无新鲜出血灶发现，且可增强HSP70与TGFβ-1的表达。(4) 水蛭提取液可增加血肿周围脑组织tPA含量、提高其活性，促进鼠血肿周围脑组织tPA mRNA表达，增强tPA免疫表达，升高血浆与血肿周围脑组织D-D含量(但以后者为主)，延长反映内源性凝血途径的aPTT，而不影响PAI-1含量与活性或mRNA表达，不影响反映外源性凝血途径的血浆PT，Fbg也不受影响。(5) 一定浓度范围(1~100U/ml)的凝血酶对As有毒性作用，且随凝血酶剂量的增大，它对As的毒性作用相应增加。(6) 水蛭提取液在一定浓度范围内(0.25~1mg/μl)可促进BMEC和As的生长，有剂量依赖关系。它能促进培养的大鼠大脑皮质BMEC分泌tPA，同时提高其活性，促进tPA mRNA的表达及tPA免疫活性表达，且呈剂量依赖性表达增强，对PAI-1的含量、活性及mRNA的表达无影响。(7) 一定浓度范围内(0.25~4mg/μl)的水蛭提取液能明显减轻10U/ml凝血酶对As的毒性作用，且随水蛭提取液浓度的增大，保护作用增强，还可促进As表达HSP70和TGFβ-1。结论：(1) 大鼠脑内血肿周围局部纤溶活性的升高有利于促进血肿的自然吸收。(2) 水蛭提取液能促进大鼠脑内血肿的吸收，同时能缩小血肿周围缺血范围并减轻脑水肿，对ICH后神经功能的恢复有明显的促进作用，而不引起出血并发症。(3) 可能的作用机制为：通过对tPA的转录、翻译及合成蛋白的加工修饰来激活内源性纤溶系统，促进胶质和内皮细胞增生，增强HSP70和TGFβ-1表达，对PAI-1无影响
Background and Purpose:It has been believed that it is a critical management to promote intracerebral hematoma to absorb faster, to control the brain edema and ameliorate the injured tissues around the hematoma after intracerebral hemorrhage (ICH) up to now. However, there is few method available to treat the hematoma itself. It is even short of efficacious medicine in promoting the hematoma to liquidize and absorb, except for extracting the hematoma by surgery. Therefore, we referred to the previous therapy studies of ICH in experimental and clinical aspects with hirudo (leech) and its polypharmacy. Our aims of this study is to explore the native absorption mechanisms of experimental intracerebral hematoma in Wistar rats, mainly to investigate the effect of hirudo extract liquor (HEL) on intracerebral hematoma in rats and its potential pharmaceutical mechanisms from the body, tissues, cells to molecular biology level by the method of combined with in vivo and in vitro, also investigate related complications after HEL therapy, trying to provide more experimental bases for its clinical use in future.
Methods: We established the experimental ICH model in rats by stereotaxical injecting quantitative collagenase(0.7U collagenaseⅦ) into their left caudate nuclei. In vitro, brain microvascular endothelial cells (BMEC) and astrocytes (As) from Wistar rat cerebral cortex were cultured respectively. In the present study, hematoma volume, infarction volume in perihematoma tissues (PHT), neurological severity scores, brain water content (BWC), biochemical index, including tissue-type
plasminogen activator (tPA) and plasminogen activator inhibitor (PAI) activity, and tPA, PAI-1and D-dimer (D-D) contents in plasma, PHT and cultured supernatants, as well as variation of semi-quantification of tPA, PAI-1 mRNA levels by RT-PCR in PHT and in cultured rat BMEC, were determined respectively. Fbg, PT, aPTT levels in plasma were simultaneously determined too. Histopathological changes including light microscope, electronic microscope and angiography were also observed. At the same time, the effects of HEL on them were delineated. BWC was calculated by drying-weighing method. Neurological deficits were examined with modified Bederson's method, infarction area with TTC staining. Activities of tPA, PAI were measured by colorimetric assay. Contents of tPA, PAI-1 and D-D were respectively determined by using specific ELISA. Local capillaries were observed by angiography with ink-dextran infusion. Expression of HSP70,TGFβ-1 and tPA protein in PHT,BMEC and As was investigated by immunohistochemistry.Cell activity or injury was assayed by measuring mitrochondrial function (MTT assay). Cell morphology under the inverted phase contrast microscope was observed and cell activity with MTT assay was measured after exposing BMEC to a selected concentration of HEL ranging from 0.0625 to 8 mg/μl. The survival of As was investigated in vitro by exposing the cells to a selected concentration of thrombin ranging from 0.1 to 100U/ml and of HEL ranging from 0.25 to 4 mg/μl by observing cell morphology and measuring the lactate dehydrogenase (LDH) from damaged cells, a marker of cell death.
Results:(1) There was ischemic damage in PHT during the acute phase of experimental ICH. (2) With the time past, intracerebral hematoma became smaller and smaller, but it was remarkably small till the 10th day, leaving incomplete absorption in some rats till the 14th day. The tPA amount, activity and its mRNA expression in PHT were increased, aPTT lengthened on the 3rd day and the 6th day after experimental ICH, showing dose-dependent in the experiment. However, PAI-1 amount, PAI relative activity and PAI-1 mRNA expression were not obviously depressed. (3) HEL could quicken intracerebral hematoma in rats to liquidize and absorb, significantly decreasing the hematoma volume and ischemic area on the 6th and 10th day, as well as
reduce brain edema, markedly decreasing BWC both at 48 hours and on the 4th day. In addition, the recovery of neurological
目前认为治疗脑出血(intracerebral hemorrhage,ICH)的根本措施就是促进脑内血肿的尽早吸收、控制脑水肿和减轻或逆转血肿周围组织的损伤。但针对血肿本身的治疗研究不多，临床上除手术清除血肿外，内科治疗尚缺乏促进血肿迅速溶解吸收的有效药物，基本上属对症治疗。因此，寻找既能促进脑内血肿迅速溶解，又能促进胶质增生，从而加快坏死组织的清除、分泌多种神经营养因子对血肿周围神经组织损伤有干预作用的药物，来延长受损神经元的存活和加快自身修复，是ICH急性期非手术治疗脑内血肿、保护因缺血引起的神经元损伤，降低死亡率、减少致残率的关键。已有人用水蛭或经炮制过的全水蛭粗提品对ICH或脑梗死进行过实验和临床研究，发现它有清除自由基、降低血浆内皮素含量、增加血浆降钙素基因相关肽含量、抗凋亡、改善脑血流量等功效。目前对缺血性心脑血管病的治疗已成共识，也有少数全水蛭中药复合物治疗ICH的报道，提示它有缩短血肿吸收时间的作用，亦未发现该药引起凝血机制正常者的再出血副作用。但他们都未从水蛭素是凝血酶最强的特异性抑制剂这一最重要的药理性质出发，考察水蛭提取液对凝血、纤溶平衡的调节来探索其消除血肿，加快自身修复的可能机制。因此，我们的研究目的就是结合体内与体外实验，从整体、组织、细胞到分子生物学水平来研究脑内血肿自然吸收的规律与机制，试图证实水蛭提取液对急性期实验性脑内血肿的吸收有促进作用，并揭示其可能的作用机制，观察其毒副作用，为其临床治疗脑内血肿提供更多的实验依据。方法：本研究拟采用体内实验即胶原酶诱导大鼠ICH模型，体外实验即大鼠大脑皮质BMEC和As培养为实验模型，选多项观察指标包括实验鼠神经功能缺损体征评分；病理改变(普通光镜、电镜与脑微血管造影)；脑水含量、血肿容积与血肿周围缺血体积测量；生化指标(tPA、PAI-1、D-D、Fbg、PT、aPTT)检测；免疫组化及RT-PCR检测。结果：(1) 大鼠 ICH后急性期血肿周围存在缺血性损害。(2) 脑内血肿随时间推移逐渐缩小，但需10d方能明显缩小，14d时仍有部分大鼠血肿尚未完全
吸收。ICH后第3d、第6d血肿周围脑组织tPA含量、活性与mRNA表达增加及aPTT延长，且呈时间依赖性。PAI-1含量与活性或mRNA表达无明显抑制。(3) 水蛭提取液能促进脑内血肿的吸收，明显吸收发生于治疗后第6d，同时能缩小血肿周围缺血范围、降低脑水含量。对ICH后神经功能的恢复有明显的促进作用。病理检查发现它能促进病灶周围血管内皮细胞、毛细血管和胶质细胞增生，无新鲜出血灶发现，且可增强HSP70与TGFβ-1的表达。(4) 水蛭提取液可增加血肿周围脑组织tPA含量、提高其活性，促进鼠血肿周围脑组织tPA mRNA表达，增强tPA免疫表达，升高血浆与血肿周围脑组织D-D含量(但以后者为主)，延长反映内源性凝血途径的aPTT，而不影响PAI-1含量与活性或mRNA表达，不影响反映外源性凝血途径的血浆PT，Fbg也不受影响。(5) 一定浓度范围(1~100U/ml)的凝血酶对As有毒性作用，且随凝血酶剂量的增大，它对As的毒性作用相应增加。(6) 水蛭提取液在一定浓度范围内(0.25~1mg/μl)可促进BMEC和As的生长，有剂量依赖关系。它能促进培养的大鼠大脑皮质BMEC分泌tPA，同时提高其活性，促进tPA mRNA的表达及tPA免疫活性表达，且呈剂量依赖性表达增强，对PAI-1的含量、活性及mRNA的表达无影响。(7) 一定浓度范围内(0.25~4mg/μl)的水蛭提取液能明显减轻10U/ml凝血酶对As的毒性作用，且随水蛭提取液浓度的增大，保护作用增强，还可促进As表达HSP70和TGFβ-1。结论：(1) 大鼠脑内血肿周围局部纤溶活性的升高有利于促进血肿的自然吸收。(2) 水蛭提取液能促进大鼠脑内血肿的吸收，同时能缩小血肿周围缺血范围并减轻脑水肿，对ICH后神经功能的恢复有明显的促进作用，而不引起出血并发症。(3) 可能的作用机制为：通过对tPA的转录、翻译及合成蛋白的加工修饰来激活内源性纤溶系统，促进胶质和内皮细胞增生，增强HSP70和TGFβ-1表达，对PAI-1无影响
Background and Purpose:It has been believed that it is a critical management to promote intracerebral hematoma to absorb faster, to control the brain edema and ameliorate the injured tissues around the hematoma after intracerebral hemorrhage (ICH) up to now. However, there is few method available to treat the hematoma itself. It is even short of efficacious medicine in promoting the hematoma to liquidize and absorb, except for extracting the hematoma by surgery. Therefore, we referred to the previous therapy studies of ICH in experimental and clinical aspects with hirudo (leech) and its polypharmacy. Our aims of this study is to explore the native absorption mechanisms of experimental intracerebral hematoma in Wistar rats, mainly to investigate the effect of hirudo extract liquor (HEL) on intracerebral hematoma in rats and its potential pharmaceutical mechanisms from the body, tissues, cells to molecular biology level by the method of combined with in vivo and in vitro, also investigate related complications after HEL therapy, trying to provide more experimental bases for its clinical use in future.
Methods: We established the experimental ICH model in rats by stereotaxical injecting quantitative collagenase(0.7U collagenaseⅦ) into their left caudate nuclei. In vitro, brain microvascular endothelial cells (BMEC) and astrocytes (As) from Wistar rat cerebral cortex were cultured respectively. In the present study, hematoma volume, infarction volume in perihematoma tissues (PHT), neurological severity scores, brain water content (BWC), biochemical index, including tissue-type
plasminogen activator (tPA) and plasminogen activator inhibitor (PAI) activity, and tPA, PAI-1and D-dimer (D-D) contents in plasma, PHT and cultured supernatants, as well as variation of semi-quantification of tPA, PAI-1 mRNA levels by RT-PCR in PHT and in cultured rat BMEC, were determined respectively. Fbg, PT, aPTT levels in plasma were simultaneously determined too. Histopathological changes including light microscope, electronic microscope and angiography were also observed. At the same time, the effects of HEL on them were delineated. BWC was calculated by drying-weighing method. Neurological deficits were examined with modified Bederson's method, infarction area with TTC staining. Activities of tPA, PAI were measured by colorimetric assay. Contents of tPA, PAI-1 and D-D were respectively determined by using specific ELISA. Local capillaries were observed by angiography with ink-dextran infusion. Expression of HSP70,TGFβ-1 and tPA protein in PHT,BMEC and As was investigated by immunohistochemistry.Cell activity or injury was assayed by measuring mitrochondrial function (MTT assay). Cell morphology under the inverted phase contrast microscope was observed and cell activity with MTT assay was measured after exposing BMEC to a selected concentration of HEL ranging from 0.0625 to 8 mg/μl. The survival of As was investigated in vitro by exposing the cells to a selected concentration of thrombin ranging from 0.1 to 100U/ml and of HEL ranging from 0.25 to 4 mg/μl by observing cell morphology and measuring the lactate dehydrogenase (LDH) from damaged cells, a marker of cell death.
Results:(1) There was ischemic damage in PHT during the acute phase of experimental ICH. (2) With the time past, intracerebral hematoma became smaller and smaller, but it was remarkably small till the 10th day, leaving incomplete absorption in some rats till the 14th day. The tPA amount, activity and its mRNA expression in PHT were increased, aPTT lengthened on the 3rd day and the 6th day after experimental ICH, showing dose-dependent in the experiment. However, PAI-1 amount, PAI relative activity and PAI-1 mRNA expression were not obviously depressed. (3) HEL could quicken intracerebral hematoma in rats to liquidize and absorb, significantly decreasing the hematoma volume and ischemic area on the 6th and 10th day, as well as
reduce brain edema, markedly decreasing BWC both at 48 hours and on the 4th day. In addition, the recovery of neurological
引文
1 Arand AG,Sawaya R.Intraoperative,chemical hemostasis in neurosurgery. Neurosurgery,1986,18(2):223~233
2 Lee KR,Kawei N,Kim S,et al.Mechanisms of edema formation after intracerebral hemorrhage:effects of thrombin on cerebral blood flow,blood-brain-barrier permeability,and cell survival in a rat model.J Neurosurg,1997,86(2):272(278
3 Gingrich MB,Junge CE,Lyuboslavsky P,et al.Potentiation of NMDA receptor function by the serine protenase thrombin.J Neurosci,2000,20(12):4582~4596
4 Nakajima K,Kohsaka S.Functional role of moiroglia in the brain.Neurosci Res,1993,17(3):187~203
5 Kimelberg HK.Receptors on astrocytes—what possible functions.Neurochem Int,1995,26(1):27~40
6 Ren JM,Finklestein SP.Time window of infarct rduction by intravenous basic fibroblast growth factor in focal cerebral is chemia.Eur J Pharmacol,1997,327:11~16
7 Beilharz EJ,Russo VC,Butler G, et al.Co-ordinated and cellular specific induction of the components of the IGF/IGFBP axis in the rat brain following hypoxic ischemia injury.Brain Res Mol Brain Res,1998,59:119~134
8 Streit WJ,Graeber MB,Kreutzberg GW.Functional plasticity of microglia:a review.Glia,1998,1(5):301~307
9 Monreal M,Costa J,Jalva P.Pharmcological properties of hirudin and its derivatives:potential clinical advantage over heparin.Drugs Aging,1996,8(3):171~182
10 Meyer BJ,Badimon JJ,Chesebro JH,et al.Dissolution of mural thrombus by specific thrombin inhibition with r-hirudin comparison with heparin and aspirin.Circulation, 1998,97:681~685
11 Biemond BJ,Levi M,Coronel R,et al.Thrombolysis and reocclusion in experimental jugular vein and coronary artery thrombosis: effects of a plasminogen activator inhibitor type1-neutralizing monoclonal antibody.Circulation, 1995,91:1175~1181
12 Chesebro JH,Rao AK,Schwartz D,et al.Endogenous thrombolysis and recanalization of occluded aortocornonary vei grafts with recombinant hirudin in patients with unstable angina.Circulation,1994,90(suppl I):I-568.Abstract
13 张莅峡,郭育芝,刘泓等.中药水蛭中的微量元素和氨基酸的分析.沈阳医学院学报,1991,8(3):172
14 贾元印, 姚乾元,汪厚增等.水蛭中抗凝血物质的提取及其药理实验.山东医药工业,1990,9(1):1
15 陈聪聪,钱宗云,陈连林,等.几种动物类中药对纤维蛋白溶解系统的实验观察.浙江中医杂志,1987,22(7):310
16 陈贵海,吴强,尹世杰,等.水蛭提取液对脑出血治疗作用的实验研究.中国危重病急救医学,1998,10(6):329~33
17 杨文清,施新猷,王四旺,等.光化学诱导大鼠大脑脑血栓及水蛭提取物的治疗作用.上海实验动物科学,1999,19(4):197~200
18 杨文清,王四旺,谢艳华,等.水蛭提取物对大鼠脑血栓后脑组织MDA、SOD和NO含量的影响.第四军医大学学报,2001,22(2):116~118
19 朱冬胜,徐敏华,危洪昌,等.中风系列制剂治疗急性脑出血的临床与实验研究.中国中西医
结合急救杂志,2000,7(3):133~137
20谢艳华,王四旺,王跃民,等.水蛭提取物对犬脑血流量的影响.第四军医大学学报,1997,18(6):518~521
21曹翠丽,马常升,马文领,等.中药水蛭对缺血/再灌注后脑细胞的抗凋亡作用.河北医科大学学报,2000,21(4):193~195
22董少龙.水蛭注射液对高血压性脑出血颅内血肿吸收的影响.广西中医药,1998,21(3):4~6
23 许志强,蒋晓江,陈曼娥.脉血康胶囊治疗脑出血30例疗效观察.中医杂志,2000,41(12):726~727
24 Rosenberg GA,Mun-Bryce S,Wesley M,et al.Collagenase-induced intracerebral hemorrhage in rats.Stroke,1990,21:801~807
25 包新民,舒斯云.大鼠脑立体定位图谱.北京:人民卫生出版社,1991
26 Harris TJ,Patel T,Marston FA,et al.Cloning of cDNA coding for human tissue-type plasminogen activator and its expression in Escherichia coli.Mol Biol Med,1986,3 (3):279~292
27 Feng P,Ohlsson M,Ny T.The structure of the TATA-less rat tissue-type plasminogen activator gene.Species-specific sequence divergences in the promoter predict differences in regulation of gene expression.J Biol Chem.1990,265(4):2022~7
28 Ginsburg D,Zeheb R,Yang AY,et al.cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.J Clin Invest, 1986,78 (6):1673~1680
29 Zeheb R,Gelehrter TD.Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1.Gene,1988,73 (2):459~468
30 马骉,舒宁,谢昌平,等.RT-PCR法检测大鼠脑组织及C6细胞中β-APP的表达. 细胞生物学,1997,19(2):92~94
31 Lowry OH,Rosenbrough NT,Farr LA,et al.Protein measurement with the Folin phenol reagent.J Biol Chem,1951,193:265~275
32 Bederson JB,Pitts LH,Nishimura MC,et al.Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.Stroke,1986,17:472~476
33 Kuluz JW,Prado RT,Dietrich D,et al.The effect of nitric oxide synthase inhibition on infarct volume after reversible focal ischemia in conscious rats.Stroke,1993,24:2033
34 Gurevich B,et al. J Neurosurg,1998,88:1066~1074
35 Gotoh O,Sano A,Koide T,et al.Ischemic brain edema following occlusion of the middle cerebral artery in the rat.Stroke,1985,16(1):101~109
36刘凤英.微血管墨汁灌注透明标本制作方法.见:田牛等著,微循环方法学(增订版).北京:原子能出版社,1993:69~74
37 Deinsberger W,Vogel J,Kuschinsky W,et al.Experimental intracerebral hemorrhage:description of a double injection model in rats.Neurol Res,1996,18(10):475~477
38 Lopez VE,Hemandez LA,Calandre L,et al.Time window for clinical effectiveness of mass evaculation in a rat balloon model mimicking an intraparenchymatous hematoma.J Neurol Sci,2000,174(1):40~46
39 Kazui S,Naritomi H,Yamamoto H,et al.Enlargement of spontaneous intracerebral hemorrhage incidence and cource.Stroke,1996,27:1783~1787
40 郑伟,谭琦.脑血康治疗高血压脑出血附148例临床疗效分析.中国中医急 症,1994,3(4):160~162
41谢道珍,周绍华,孙怡,等.脑血康治疗高血压性脑出血的临床与实验研究.中西医结合杂志,1988,8(6):341~343
42 Pang L,Ye W,Che XM,et al.Reduction of inflammatory response in the mouse brain with adenoviral mediated transforming growth factor-β1 expression.Stroke,2001,32(2):544~552
43 Henrich-Noack P,Prehn JH,Krieglstein J.TGF-beta 1 protects hippocampal neurons against degeneration caused by transient global ischemia.Dose-response relationship and potential neuroprotective mechanisms.Stroke,1996,27(9):1609
44 Krupinski J,Kumar P,Kumar S,et al.Increased expression of TGF-beta 1 in brain tissue after ischemic stroke in humans.Stroke,1996,27(5):852
45 Bukau B,Horwich Q.The Hsp70 and Hsp60 Chaperone machines.Cell,1998,92:351~366.
46 Voisine C,Craig EA,Zufall N,et al.The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70.Cell,1999, 28,97(5):565~74.
47 Katayama T,Imaizumi K,Tsuda M,et al.Expression of an ADP-ribosylation factor like gene,ARF4L,is induced after transient forebrain ischemia in the gerbil. Brain Res Mol Brain Res.1998,56(1~2):66~75.
48 Tomimoto H,Takemoto O,Akiguchi I,et al.Immunoelectron microscopic study of c-Fos， c-Jun and heat shock protein after transient cerebral ischemia in gerbils.Acta Neuropathol Berl.1999，97(1):22~30.
49 Mosser DD.Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis.Mel Cell Biol,1997,17:5317
50 Nath FP,Jenkins A,Mendelow AD,et al.Early hemodynamic changes in experimental intracerebral hemorrhage. J Neurosurg,1986,65(4):697(703
51 Yang GY,Betz AL,Chenevert TL,et al.Experimental intracerebral hemorrhage: relationship between brain edema,blood flow, and blood-brain-barrier permeability in rats.J Neurosurg,1994,81(1):93(102
52 Mayer SA,Lignelli A,Tink ME,et al.Perilesional blood flow and edema formation in acute intrcrerebral hemorrhage:a SPECT study.Stroke,1998,29(10):1791～17998
53 Villar-Cordova C,Krieger D,Mullani N,et al.Hypometabolism and ischemic penumbra surrounding intrcrerebral hemorrhage in humans demonstrated by Positron Emission Tomography.Stroke,1997,28(2):254
54 Zlokovic BV.Expression of tissue plasminogen activator in cerebral capillaries:possible fibrinolytic function of the blood-brain barrier. Neurosurgery,1995,37:955
55 Sappino AP,Madani R,Huarte J,et al.Extracellular proteolysis in the adult murine brain.J Clin Invest,1993,92:679
56 Inagami T,Naruse M,Richard H.Endothelium as an endocrine organ.Annu Rev Physiol，1995,57:171~189
57 Gordon EL,Danielsson PE,Hguyen TS,et al.A comparision of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells.In Vitro Cell Dev Biol,1991,27A:312~326
58 Brendel K,Meezan E,Carlson EC.Isolated brain microvessels:a purified,metabolically active preparation from bovine cerebral cortex.Science,1974,185:953~955
59 Browman PD,Betz AL,Diane AR,et al.Primary culture of capillary endothelium from rat brain.In Vitro,1981,17(4):253~262
60 Chao CC,Hu SX,Sheng WS,et al.Cytokine-stimulated astrocytes damage human neurous via a nitric oxide mechanism.Glia,1996,16:276~284.
61 McCarthy KD,Vellis JD.Preparation of separate astroglial and oligdendroglial cell cultures from rat cerebral tissue.J Cell Biol,1980,85:890~902.
62薛庆华,郭畹华.大鼠大脑皮质星形胶质细胞的体外培养及其促进神经突起生长作用研究.神经解剖学杂志,1996,12(2):151~155
63 Kesslak JP,Nieto-Sampedro M,Globus J,et al. Transplants of purified astrocytes promote behavioral recovery after frontal cortex ablation.Exp Neurol,1986,92(2):377~90
64 Silver J.Transplantation strategies using embryonic astroglial cells to promote CNS axon
regeneration in neonatal and adult mammals.Clin Res,1988,36(3):196~9
65 Bradbury EJ,Kershaw TR,Marchbanks RM,et al.Astrocyte transplants alleviate lesion induced memory deficits independently of cholinergic recovery. Neuroscience,1995,65(4):955~72
66 Horsburgh K,Graham DI,Stewart J,et al.Influence of apoli proptein E genotype on neuronal damage and apoaaaaaaae immunoreactivity in human hippocampus following global ischemia.J Neuropathol Exp Neurol,1999,58(2);227~234
67 Koh TY,Choi DW.Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase effux assay.J Neurosci Meth,1987,20(1):83~90
68董少龙,黄立武,张茂林.水蛭提取液对实验性家兔颅内血肿的影响1.广西中医药,200， 23(2):49~50
2 Lee KR,Kawei N,Kim S,et al.Mechanisms of edema formation after intracerebral hemorrhage:effects of thrombin on cerebral blood flow,blood-brain-barrier permeability,and cell survival in a rat model.J Neurosurg,1997,86(2):272(278
3 Gingrich MB,Junge CE,Lyuboslavsky P,et al.Potentiation of NMDA receptor function by the serine protenase thrombin.J Neurosci,2000,20(12):4582~4596
4 Nakajima K,Kohsaka S.Functional role of moiroglia in the brain.Neurosci Res,1993,17(3):187~203
5 Kimelberg HK.Receptors on astrocytes—what possible functions.Neurochem Int,1995,26(1):27~40
6 Ren JM,Finklestein SP.Time window of infarct rduction by intravenous basic fibroblast growth factor in focal cerebral is chemia.Eur J Pharmacol,1997,327:11~16
7 Beilharz EJ,Russo VC,Butler G, et al.Co-ordinated and cellular specific induction of the components of the IGF/IGFBP axis in the rat brain following hypoxic ischemia injury.Brain Res Mol Brain Res,1998,59:119~134
8 Streit WJ,Graeber MB,Kreutzberg GW.Functional plasticity of microglia:a review.Glia,1998,1(5):301~307
9 Monreal M,Costa J,Jalva P.Pharmcological properties of hirudin and its derivatives:potential clinical advantage over heparin.Drugs Aging,1996,8(3):171~182
10 Meyer BJ,Badimon JJ,Chesebro JH,et al.Dissolution of mural thrombus by specific thrombin inhibition with r-hirudin comparison with heparin and aspirin.Circulation, 1998,97:681~685
11 Biemond BJ,Levi M,Coronel R,et al.Thrombolysis and reocclusion in experimental jugular vein and coronary artery thrombosis: effects of a plasminogen activator inhibitor type1-neutralizing monoclonal antibody.Circulation, 1995,91:1175~1181
12 Chesebro JH,Rao AK,Schwartz D,et al.Endogenous thrombolysis and recanalization of occluded aortocornonary vei grafts with recombinant hirudin in patients with unstable angina.Circulation,1994,90(suppl I):I-568.Abstract
13 张莅峡,郭育芝,刘泓等.中药水蛭中的微量元素和氨基酸的分析.沈阳医学院学报,1991,8(3):172
14 贾元印, 姚乾元,汪厚增等.水蛭中抗凝血物质的提取及其药理实验.山东医药工业,1990,9(1):1
15 陈聪聪,钱宗云,陈连林,等.几种动物类中药对纤维蛋白溶解系统的实验观察.浙江中医杂志,1987,22(7):310
16 陈贵海,吴强,尹世杰,等.水蛭提取液对脑出血治疗作用的实验研究.中国危重病急救医学,1998,10(6):329~33
17 杨文清,施新猷,王四旺,等.光化学诱导大鼠大脑脑血栓及水蛭提取物的治疗作用.上海实验动物科学,1999,19(4):197~200
18 杨文清,王四旺,谢艳华,等.水蛭提取物对大鼠脑血栓后脑组织MDA、SOD和NO含量的影响.第四军医大学学报,2001,22(2):116~118
19 朱冬胜,徐敏华,危洪昌,等.中风系列制剂治疗急性脑出血的临床与实验研究.中国中西医
结合急救杂志,2000,7(3):133~137
20谢艳华,王四旺,王跃民,等.水蛭提取物对犬脑血流量的影响.第四军医大学学报,1997,18(6):518~521
21曹翠丽,马常升,马文领,等.中药水蛭对缺血/再灌注后脑细胞的抗凋亡作用.河北医科大学学报,2000,21(4):193~195
22董少龙.水蛭注射液对高血压性脑出血颅内血肿吸收的影响.广西中医药,1998,21(3):4~6
23 许志强,蒋晓江,陈曼娥.脉血康胶囊治疗脑出血30例疗效观察.中医杂志,2000,41(12):726~727
24 Rosenberg GA,Mun-Bryce S,Wesley M,et al.Collagenase-induced intracerebral hemorrhage in rats.Stroke,1990,21:801~807
25 包新民,舒斯云.大鼠脑立体定位图谱.北京:人民卫生出版社,1991
26 Harris TJ,Patel T,Marston FA,et al.Cloning of cDNA coding for human tissue-type plasminogen activator and its expression in Escherichia coli.Mol Biol Med,1986,3 (3):279~292
27 Feng P,Ohlsson M,Ny T.The structure of the TATA-less rat tissue-type plasminogen activator gene.Species-specific sequence divergences in the promoter predict differences in regulation of gene expression.J Biol Chem.1990,265(4):2022~7
28 Ginsburg D,Zeheb R,Yang AY,et al.cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.J Clin Invest, 1986,78 (6):1673~1680
29 Zeheb R,Gelehrter TD.Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1.Gene,1988,73 (2):459~468
30 马骉,舒宁,谢昌平,等.RT-PCR法检测大鼠脑组织及C6细胞中β-APP的表达. 细胞生物学,1997,19(2):92~94
31 Lowry OH,Rosenbrough NT,Farr LA,et al.Protein measurement with the Folin phenol reagent.J Biol Chem,1951,193:265~275
32 Bederson JB,Pitts LH,Nishimura MC,et al.Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.Stroke,1986,17:472~476
33 Kuluz JW,Prado RT,Dietrich D,et al.The effect of nitric oxide synthase inhibition on infarct volume after reversible focal ischemia in conscious rats.Stroke,1993,24:2033
34 Gurevich B,et al. J Neurosurg,1998,88:1066~1074
35 Gotoh O,Sano A,Koide T,et al.Ischemic brain edema following occlusion of the middle cerebral artery in the rat.Stroke,1985,16(1):101~109
36刘凤英.微血管墨汁灌注透明标本制作方法.见:田牛等著,微循环方法学(增订版).北京:原子能出版社,1993:69~74
37 Deinsberger W,Vogel J,Kuschinsky W,et al.Experimental intracerebral hemorrhage:description of a double injection model in rats.Neurol Res,1996,18(10):475~477
38 Lopez VE,Hemandez LA,Calandre L,et al.Time window for clinical effectiveness of mass evaculation in a rat balloon model mimicking an intraparenchymatous hematoma.J Neurol Sci,2000,174(1):40~46
39 Kazui S,Naritomi H,Yamamoto H,et al.Enlargement of spontaneous intracerebral hemorrhage incidence and cource.Stroke,1996,27:1783~1787
40 郑伟,谭琦.脑血康治疗高血压脑出血附148例临床疗效分析.中国中医急 症,1994,3(4):160~162
41谢道珍,周绍华,孙怡,等.脑血康治疗高血压性脑出血的临床与实验研究.中西医结合杂志,1988,8(6):341~343
42 Pang L,Ye W,Che XM,et al.Reduction of inflammatory response in the mouse brain with adenoviral mediated transforming growth factor-β1 expression.Stroke,2001,32(2):544~552
43 Henrich-Noack P,Prehn JH,Krieglstein J.TGF-beta 1 protects hippocampal neurons against degeneration caused by transient global ischemia.Dose-response relationship and potential neuroprotective mechanisms.Stroke,1996,27(9):1609
44 Krupinski J,Kumar P,Kumar S,et al.Increased expression of TGF-beta 1 in brain tissue after ischemic stroke in humans.Stroke,1996,27(5):852
45 Bukau B,Horwich Q.The Hsp70 and Hsp60 Chaperone machines.Cell,1998,92:351~366.
46 Voisine C,Craig EA,Zufall N,et al.The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70.Cell,1999, 28,97(5):565~74.
47 Katayama T,Imaizumi K,Tsuda M,et al.Expression of an ADP-ribosylation factor like gene,ARF4L,is induced after transient forebrain ischemia in the gerbil. Brain Res Mol Brain Res.1998,56(1~2):66~75.
48 Tomimoto H,Takemoto O,Akiguchi I,et al.Immunoelectron microscopic study of c-Fos， c-Jun and heat shock protein after transient cerebral ischemia in gerbils.Acta Neuropathol Berl.1999，97(1):22~30.
49 Mosser DD.Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis.Mel Cell Biol,1997,17:5317
50 Nath FP,Jenkins A,Mendelow AD,et al.Early hemodynamic changes in experimental intracerebral hemorrhage. J Neurosurg,1986,65(4):697(703
51 Yang GY,Betz AL,Chenevert TL,et al.Experimental intracerebral hemorrhage: relationship between brain edema,blood flow, and blood-brain-barrier permeability in rats.J Neurosurg,1994,81(1):93(102
52 Mayer SA,Lignelli A,Tink ME,et al.Perilesional blood flow and edema formation in acute intrcrerebral hemorrhage:a SPECT study.Stroke,1998,29(10):1791～17998
53 Villar-Cordova C,Krieger D,Mullani N,et al.Hypometabolism and ischemic penumbra surrounding intrcrerebral hemorrhage in humans demonstrated by Positron Emission Tomography.Stroke,1997,28(2):254
54 Zlokovic BV.Expression of tissue plasminogen activator in cerebral capillaries:possible fibrinolytic function of the blood-brain barrier. Neurosurgery,1995,37:955
55 Sappino AP,Madani R,Huarte J,et al.Extracellular proteolysis in the adult murine brain.J Clin Invest,1993,92:679
56 Inagami T,Naruse M,Richard H.Endothelium as an endocrine organ.Annu Rev Physiol，1995,57:171~189
57 Gordon EL,Danielsson PE,Hguyen TS,et al.A comparision of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells.In Vitro Cell Dev Biol,1991,27A:312~326
58 Brendel K,Meezan E,Carlson EC.Isolated brain microvessels:a purified,metabolically active preparation from bovine cerebral cortex.Science,1974,185:953~955
59 Browman PD,Betz AL,Diane AR,et al.Primary culture of capillary endothelium from rat brain.In Vitro,1981,17(4):253~262
60 Chao CC,Hu SX,Sheng WS,et al.Cytokine-stimulated astrocytes damage human neurous via a nitric oxide mechanism.Glia,1996,16:276~284.
61 McCarthy KD,Vellis JD.Preparation of separate astroglial and oligdendroglial cell cultures from rat cerebral tissue.J Cell Biol,1980,85:890~902.
62薛庆华,郭畹华.大鼠大脑皮质星形胶质细胞的体外培养及其促进神经突起生长作用研究.神经解剖学杂志,1996,12(2):151~155
63 Kesslak JP,Nieto-Sampedro M,Globus J,et al. Transplants of purified astrocytes promote behavioral recovery after frontal cortex ablation.Exp Neurol,1986,92(2):377~90
64 Silver J.Transplantation strategies using embryonic astroglial cells to promote CNS axon
regeneration in neonatal and adult mammals.Clin Res,1988,36(3):196~9
65 Bradbury EJ,Kershaw TR,Marchbanks RM,et al.Astrocyte transplants alleviate lesion induced memory deficits independently of cholinergic recovery. Neuroscience,1995,65(4):955~72
66 Horsburgh K,Graham DI,Stewart J,et al.Influence of apoli proptein E genotype on neuronal damage and apoaaaaaaae immunoreactivity in human hippocampus following global ischemia.J Neuropathol Exp Neurol,1999,58(2);227~234
67 Koh TY,Choi DW.Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase effux assay.J Neurosci Meth,1987,20(1):83~90
68董少龙,黄立武,张茂林.水蛭提取液对实验性家兔颅内血肿的影响1.广西中医药,200， 23(2):49~50