【作者】 林坚；

【导师】 刘伟国；

【作者基本信息】 浙江大学 ， 神经外科， 2004， 硕士

【摘要】 脑出血在临床上非常常见，其死亡率、致残率均较高。然而，在许多的动物实验和临床研究中均发现，中小量的出血本身并不引起颅内压显著升高，因此，血肿周围组织水肿及继发性神经元损伤是引发严重后果的原因之一。但其发生发展机制仍未阐明，临床治疗也缺乏有效方法。脑出血后脑水肿的产生机制近年的研究可归纳如下几点：血肿周围组织缺血缺氧、凝血酶作用、血肿成分的影响、免疫及炎症反应。近年来免疫炎症反应在脑损伤中的作用逐渐受到重视。许多文献证实在脑损伤后炎性细胞产生及激活，释放大量炎性细胞因子，如TNF-α、IL-8、IL-6等。它们是补体蛋白生物学的介质。而补体系统是炎症反应及体液免疫的重要组成部分，在机体起防御反应和免疫调节作用，也可介导免疫病理的损伤性反应。近年来国内外资料表明实验和临床研究发现补体系统在脑损伤的炎症反应中扮演重要的角色。Davis等在1992年对脑梗死患者的研究中，发现脑梗死患者血清C4水平降低，而膜攻击复合物MAC水平明显高于正常。1998年浙江人学硕十研究生学位论文Lindsberg等在人类血脑屏障与中枢神经系统补体活性的研究中发现，人脑组织在发生脑梗死区及周围的水肿带区有不同程度的C9沉积。2001年GuohuaXi等研究发现，补体系统耗竭的大鼠，在发生脑出血时，其脑水肿程度卜降，炎症介质浸润减少，提示补体系统在出血性脑水肿的发生中起一定作用。 眼镜蛇毒因子(CvF)是从眼镜蛇毒液中提取的一个无毒的糖蛋白。它能裂解C3和CS，是补体的旁路激活物。CvF既能激活补体又能最终耗竭补体，是研究补体较理想的工具。查阅国内外相关资料发现，对补体系统与神经系统关系的研究不多，且多限于对正常动物的补体系统耗竭以后再作相关性研究，如补体系统耗竭与脑梗塞的关系，补体系统耗竭对大面积烧伤大鼠的研究等。但对于正常机体脑出血后补体系统过度激活对脑水肿的变化的研究都少见相关报道。本实验通过CVF激活补体系统，观察兔脑出血.急性期脑水含量的变化及对肿瘤坏死因子(TNF一。)，神经元特异性烯醇化酶(NSE)不同时点浓度变化，研究补体系统对脑出血后脑水肿及TNF、NSE的动态变化，为临床治疗出血性脑水肿提供可能的途径。 材料与方法1.1、材料及分组 实验用兔由温州医学院动物实验中心提供的日本大耳兔56只，均为雄性，体重2300士300克。随机分为四组，一组为对照组20只，二组为实验组20只，三组为空白组8只，四组为标准组8只。浙江大学硕十研究生学位论文2、实验步骤及方法 对照组日本大耳兔耳背动脉取血2ml于玻璃试管内，取上清液送检， 待测补体C3、TNF、NSE。用经醉素处理的lml注射器抽动脉血lml备用。水合氯醛针3ml/kg腹腔内注射麻醉后，固定兔头部，在左侧眼中点与矢状缝连线上，旁开矢状缝约0.scm，用颅锥锥穿颅骨，将lml注射器针头插入1.scm，快速将经配素处理的0.sml动脉血注入兔脑内，等待5分钟后拔除注射器。6小时、24小时取血待测TNF、NSE。48小时取血待测补体C3，TNF，NSE。取血后马上断头取兔完整大脑，其中2只在10cy0福尔马林液中固定，备做常规蜡块。余18只大脑被切开左右半球，马上用电子分析天秤称取半球大脑湿重。然后放在100℃烘箱中，24小时后取出称得干重。以(湿一一曰/湿作为脑水的含量。实验组兔脑内注入动脉血，并按0.smg/kg腹腔内注射含CVF的生理盐水约5耐;空白组兔脑内不注入动脉血，但腹腔注射CVF。标准组脑内不注入动脉血，腹腔注射5ml生理盐水。取血一时点、检测指标四组均相同。1 .3、实验设备与试剂 补体c3试剂盒由杭州惠利医疗科技开发有限公司提供。NsE试剂盒和检测仪Eleesys 2010型由瑞士罗氏公司提供，TNF一。试剂盒和IMMULITE;检测仪由美国DPC公司提供。分光光度计离心机、冷冻箱均由温州医学院附属第二医院检验科提供。烘烤箱、兔固定器、电子天杆由温州医学院动物实验中心提供。眼镜蛇毒粉剂购自广西医科大学蛇毒研究所。1.4、统计方法浙江人学硕士研究生学位论文 全部数据采用SPSS一f。犷windows软件处理。对照组、实验组、空白组、标准组Oh、6h、24h、48h血浆中TNF、NSE;Oh、48h血浆中补体C3;48小时后左右脑(湿一干)/湿比重进行统计分析。组间比较用方差分析，组间比较用方差分析或独立样本t检验。今士二目果指标一:补体C3 吐组在O时点比较F=0 .352，P>0 .05，表明补体C3含量差别无统计学意义。在48时点时F二82.1 18，P<0.01，表明各组C3含量差别有统计学意义。对照到_!与实验组、空白组比较均P<0 .01，表明在铭小时点时补体C3的差别有统计学意义;实验组、空白组与标准组比较，均P<0.01，表明在谨8时点时补体C3的差别有统计学意义。实验组与空白组组内的O时点、48时点补体C3比较P< 0.001，表明补体C3的差别有统计学意义。指标二:NSE 性组在O时点NSE比较F=0 .103，P>0.05，表明各组在O时点NSE差别在统计学上无意义。在6时点时，24、48时点，对照组与空白组、标更多还原

【Abstract】 Cerebral hemorrhage is a very common clinical disease with high mortality and morbidity rate, however, both many animal experiments and clinical studies found that little even middle level hemorrhage itself did not increase the intracranial pressure, thus, the tissue edema around the hemorrhage and secondary neuron injuries may be one of the reasons for the severe sequence. Due to the uncertainty of the real mechanism of the occurrence and progress of the disease, the effective treatment is by far not available. The possible mechanism of the cerebral edema secondary to .cerebral hemorrhage within reach till now may be summarized as follows: the tissue rschemia and anoxia around the hemorrhage; the action of the thrombin;the effect of component of the hematoma; immune and inflammatory reaction. The role of the immune inflammatory reaction on the brain injury has been gradually paid attention to in recent years and many literatures confirm that brain injury may generate and activate the inflammatory cells which can release plenty of inflammatory cytokines such as TNF-α , IL-8, IL-6 etc. belonging to the medium of complement protein families. As we all know, complement system is the important component of the inflammatory reaction and humoral immunity which stands immune defence and immunoloregulation and may mediate the immunopathological destructive reaction. In recent years many domestic and foreign experimental and clinical data both manifest that complement system plays an important role in the inflammatory reaction after brain injury. The study of Davis et al found that the C4 level decreased in the cerebral infarction patients while the level of membrane attack complex (MAC) was obviously higher than that of. the normal in 1992 and the research of Lindsberg et al on the complement activation of the human blood brain barrier and central nervous system also verified that there was different level of C9 deposition in the cerebral infraction area and the surrounding edema zone in human brain tissue in 1998. Guohuaxi’ s study also found the extent of cerebral edema declined and inflammatory infiltrationreduced when cerebral hemorrhage occurred in the complement system exhausted rat model and the result induced that the complement system may contribute to reducing the hemorrhagic cerebral edema to some extent.The cobra venom factor(CVF) is a nonvenomous glycoprotein extracted from the cobra venom which can split C3 and C5 activated by the alternative complement pathway. The CVF can activate the complement and exhaust it at last as well and is an ideal tool to study the complement. All the data available on the study of the relationship of the complement system and nervous system is rare and the related research are all confined to the study of the normal animal after complement system exhausted, such as the relationship between the complement system exhausted and cerebral infarction, the study of complement system exhausted in a large area burned rat etc. while the report of the effect of excessive activation of the complement system on cerebral edema is few. This experiment is toobserve the change of the water content in the brain tissue after acute cerebral hemorrhage and the variation of the concentration of Tumor necrosis factor (TNF-α) and Neuron-specific enolase (NSE) in different observation time by activating the complement system through CVF in rabbits and study the dynamic changes of the complementsystem on the cerebral edema after cerebral hemorrhage and the concentration of the TNF and NSE so as to find a practicable way to treat the hemorrhagic cerebral edema.Materials and Methods1.1 Material and experimental groupsA total of 56 male Japanese White rabbits (weight, 2300 + 300 g) were provided with animal-experiment center of Wenzhou Medical College. The experiments were divided into 4 groups, consisted of a control group, a CVF-treated group, a vacuity group and a standard group.1.2 Experimental procedures and MethodsJapanese White rabbit’更多还原