【作者】 吴文斌；

【导师】 胡长林；

【作者基本信息】 重庆医科大学 ， 神经病学， 2002， 博士

【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<WP=6>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无影响更多还原

【Abstract】 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<WP=8>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<WP=9>reduce brain edema, markedly decreasing BWC both at 48 hours and on the 4th day. In addition, the recovery of neurological更多还原