【作者】 邹磊；

【导师】 张世刚；

【作者基本信息】 中国医科大学 ， 外科学， 2010， 硕士

【摘要】 目的脑出血是神经系统常见病、多发病,其致残率和死亡率均很高,严重影响人类的健康和患者的生存质量。本研究利用立体定向技术向大鼠尾状核内注入50μL自体股动脉血建立脑出血模型,用脑水含量和伊文思兰染色检测BBB的通透性,HE染色观察神经损伤,通过免疫组化法检测TNF-α、NF-κB、Caspase-3表达阳性细胞在大鼠脑出血后不同时间点表达变化规律和细胞分布。该实验研究表明,细胞凋亡机制不仅参与了脑出血继发性脑损伤,而且作用时间长,损伤范围也较广。试验证明血肿近缘以坏死为主,血肿周围主要以凋亡为主,远隔部位无凋亡发生,证明了脑出血后血肿周围神经细胞损伤有凋亡机制参与。有研究发现TNF-a表达量与细胞凋亡二者NF-κB、Caspase-3呈正相关；认为TNF-a的表达是细胞凋亡触发机制之一。本试验研究旨在研究TNF-α、NF-κB和Caspase-3是否在脑出血后血肿周围组织表达及细胞分布以及NF-κB和Caspase-3的相互关系。材料与方法一、材料1、动物分组：选择健康雄性SD大鼠100只,体重250-300g,随机分成4组：正常对照组、假手术组、生理盐水对照组、脑出血实验组,生理盐水对照组和脑出血实验组又分别按6h、1d、3d、7d分为4个亚组,每个亚组n=10。2、主要仪器：动物头颅立体定位仪、电子分析天平、图像分析仪、紫外分光光度仪、微量进样器、高温干燥箱。3、主要试剂：NF-κB兔抗鼠多克隆抗体、Caspase-3兔抗鼠多克隆抗体、TNF-α兔抗鼠多克隆抗体、即用型SABC免疫组化试剂盒、DAB试剂盒、伊文思兰、甲酰胺。4、大鼠脑出血模型制作：用微量注射器抽取70uL大鼠自体股动脉血。立即将大鼠俯卧位于脑立体定位仪上,于前囟前0.2mm,中线右旁3.0mm,进针约6mm(大鼠尾状核处),将大鼠白体股动脉血50uL缓慢推注入脑。生理盐水对照组注入50uL生理盐水。假手术对照组进行手术,但不进行注射。正常对照组不予任何处置。5、标本制作：(1)每组取5只大鼠于术后相应时间点麻醉开胸,迅速暴露心脏,4%多聚甲醛灌注固定,取脑,样本置于4%多聚甲醛外固定4小时,酒精脱水,二甲苯透明,浸蜡,包埋。连续冠状切片,片厚5um,进行切片HE染色和免疫组化染色。(2)每组取5只大鼠于术后相应时间点,断头取脑后进行脑组织水含量和脑组织Evens Blue含量测定。二、检测指标1、脑组织水含量测定：于相应时间点将大鼠麻醉后断头处死,取出出血侧针孔前部脑组织,测湿重后,脑组织放入烤箱内烘干24小时后重复测定至恒重。按公式脑组织含水量(%)=(湿重-干重)／湿重×100%。以脑组织水含量代表脑水肿的程度。2、BBB通透性测定：取大鼠出血侧针孔后侧水肿区脑组织,按Belayev法使用伊文思兰(Evens blue,EB)测定BBB通透性。用EB含量(OD／mg)表示BBB通透性。3.NF-κB表达免疫组化测定：SABC法进行NF-κB蛋白、Caspase-3蛋白表达阳性的细胞测定。切片滴加50uL一抗工作液兔抗鼠多克隆NF-κB抗体(1：400),4℃过夜；然后滴加物素化山羊抗兔IgG,37℃20min；滴加SABC,37℃20min,各步骤用PBS洗3 min×3次；DAB显色剂,苏木素轻度复染,脱水透明,封片。显微图像分析系统采集图像,分析阳性细胞积分光密度。4.Caspase-3表达免疫组化测定：SABC法进行Caspase-3蛋白表达阳性的细胞测定。切片滴加50uL一抗工作液兔抗鼠多克隆Caspase-3抗体(1：150),4℃过夜；然后滴加物素化山羊抗兔IgG,37℃20min；滴加SABC,37℃20min,各步骤用PBS洗3 min×3次；DAB显色剂,苏木素轻度复染,脱水透明,封片。显微图像分析系统采集图像,分析阳性细胞积分光密度。5.TNF-α表达免疫组化测定：SABC法进行TNF-α蛋白表达阳性的细胞测定。切片滴加50uL一抗工作液兔抗鼠多克隆TNF-a抗体(1：200),4℃过夜；然后滴加物素化山羊抗兔IgG,37℃20min；滴加SABC,37℃20min,各步骤用PBS洗3 min×3次；DAB显色剂,苏木素轻度复染,脱水透明,封片。显微图像分析系统采集图像,分析阳性细胞积分光密度。三、统计分析所有数据以均数±标准差(x+SD)表示,采用SPSS16.0及Excel统计软件进行数据处理,组间比较用单因素方差分析(ANOVA),在单因素方差分析有意义的基础上再进行组间两两比较,两变量之间相关关系行Spearman相关分析,P<0.05为差异显著。结果1、脑水含量测定：大鼠实验性ICH后脑组织水含量于6h开始升高,3d达到高峰,7d时逐渐减少,但较对照组仍有显著差异(P<0.05)。2、脑组织EB含量测定：大鼠实验性ICH后脑组织EB的含量增加,6h即明显升高,3d达到最高,7d逐渐降低,但是与对照组相比仍有显著差异(P<0.05)。3、脑出血后Caspase-3蛋白表达：大鼠实验性ICH后6h在血肿周围和同侧大脑皮层有Caspase-3弱阳性表达,阳性部位主要为胞浆。1d出现大量阳性反应神经元。脑出血后3d达高峰,着色最深,呈棕褐色,并出现核阳性着色,说明Caspase-3裂解后有核转移。7d时数量减少,但积分光密度较对照组仍有显著差异(P<0.05)。4、脑出血后NF-κB表达：大鼠实验性ICH后血肿周围NF-κB阳性细胞于术后6h开始增多,1d表达继续上升,3d达高峰,此后NF-κB表达开始下降,持续到7d仍高于对照组(P<0.05)。其表达以胶质细胞为主,也有部分神经元细胞表达。阳性表达为胞核黄褐色,苏木素不能复染。血肿对侧也有少量NF-κB表达。假手术对照组和生理盐水对照组仅有极少量阳性细胞表达。5、脑出血后TNF-α的表达：大鼠实验性ICH后6h血肿周边组织TNF-a开始表达,3d达高峰,各组与对照组之间有著差异(P<0.05)。实验结果显示,血肿周围脑组织中免疫组化阳性细胞数增加,呈动态变化趋势,与光学显微镜下观察到的神经细胞水肿和周围间质组织水肿、炎性细胞浸润动态变化趋势相一致。讨论在本实验中采用向大鼠右侧尾状核内注射自体股动脉血制作脑出血模型,通过免疫组化法对NF-κB、Caspase-3 TNF-α进行检测,发现出血后的大鼠脑组织中NF-κB、Caspase-3、TNF-α均动态表达。近年来,许多实验研究都证实,细胞凋亡机制参与了脑出血后继发性损伤。在本实验中应用相邻切片分别进行Caspase-3、TNF-α和NF-κB的免疫组化染色,两者阳性细胞范围及时间变化基本一致。提示Caspase-3在脑出血后神经细胞凋亡途径中起重要调控作用。NF-κB信号通路是胞内信号转导通路中最重要的下游通路,参与炎症反应和细胞凋亡等重要的病理生理过程。本实验发现,NF-κB、Caspase-3表达量及核阳性率与TNF-a的升高呈正相关,这表明NF-κB表达的增多伴有神经细胞内Caspase-3的激活,即Caspase-3的核移位。大量文献也证实了脑出血后血肿周围存在上述反应,并认为它可能导致了脑出血后的细胞凋亡过程。由此推断NF-κB介导的Caspase-3信号途径参与脑出血后的炎症反应,并加重神经元损伤和凋亡。结论1、Caspase-3、TNF-α和NF-κB在大鼠脑出血后的血肿周围脑组织表达增高。2、Caspase-3、NF-κB、TNF-α在脑出血后脑水肿周围表达有相关性。更多还原

【Abstract】 ObjectiveCerebral hemorrhage is a common disease in neurology, which has both high disability and fatality and greatly threatens health and quality of life. In this research, Horseley-Clarke technique is used to inject 50μL of autogenetic blood from femoral artery into caudate nucleus of mouses, HE staining is chosen to observe apoptosis, immune histochemistry is used to measure the expression levels of NF-κB、Caspase-3 and TNF-a.The experimental results showed that apoptosis mechanism not only involved in cerebral hemorrhage secondary brain injury, and this role a long time, damage a wider range.Around the hematoma mainly apoptosis-based.Test to prove hematoma relatives with necrosis.Distant parts of non-apoptotic.Proved that brain nerve cells after intracerebral hemorrhage injury mechanisms involved in apoptosis.Studies have found that TNF-aexpression and apoptosis in both NF-κB, Caspase positive correlation; that TNF-aexpression is one of the mechanisms triggering apoptosis. Test purpose of the study is to explore the NF-κB and Caspase the dynamic relations in order to prove the existence of NF-κB induced Caspase-3 mediated apoptosis.Materials and methodsMaterials1. Groups100 male Sprague Dawley mouses that weigh from 250 to 300g were randomly grouped into normal control group, sham-operated control group, saline control group, experimental group. The last two groups were respectively sub-grouped by 6h, Id,3d and 7d, n=15.2. Major equipmentsExperimental cranial sterotaxic apparatus, electronic analytical balance, micro-image analysis apparatus, ultraviolet spectrometer, microsyringe, pyro-drying cabine.3. Major reagentEvens blue, formamide, NF-κB rabbit anti-mouse polyclonal antibody, Caspase-3 rabbit anti-mouse polyclonal antibody, instant SABC immunohistochemistry test kit, DAB test kit.4. Models of cerebral hemorrhageModels of slow injection with autoblood. Mouses were drawn 70uL arterial blood, put in prone position on stereotaxic apparatus, needled 0.2mm ahead of anterior fontanel,3.0mm right in median in depth of 6mm (caudate nucleus) and injected with 50uL arterial blood. Mouses in saline control group were injected with NS, Sham-operated control group without injection, while normal control group received no disposition.5. Making samples(1)5 mouses were taken from each group after the corresponding time of operation, and anesthetized, decapitated and got brain off for brain water content and permeability of BBB.(2) 5 mouses were taken from each group after the corresponding time of operation, immediately received heart exposition, infused with 4%paraform and got brains off. The taken sample were fixed in 4% paraform for 4 hours, dehydrated in alchohol, transparent by xylol, dipped in wax and embedded. Successive coronal-slice, about 5 um thick, and then stain.Measure1. Brain water contentAnterior brain tissue of needle puncture was resectioned and determined by [(wet-dry weight)/wet weight]%. With brain water content as degree of brain edema.2. Permeability of BBBPosterior lateral brain tissue of needle puncture was recetioned and assessed by Evens blue stain, as Belayev’s method. Permeability of the BBB is assessed by EB content (OD/mg).3. Measuring NF-κB protein by immunohistochemistryUse SABC to measure positive cells which express NF-κB protein. Add the slice with 50uL-antifluid(rabbit anti mouse polyclonal NF-κB antibody(1:400)), stay overnight at 4℃; add goat anti rabbit IgG, at 37℃for 20min; add SABC at 37℃ fro20min; every step washed by PBS for 3min 3 times; DAB developer, slightly restain by hematoxylin, dehydrate, transparent and mount. Use the microgram analysis system to collect images, and analyze spectrodensity of positive cells.4. Measuring Caspase-3 protein by immunohistochemistryUse SABC to measure positive cells which express Caspase-3 protein. Add slice with 50 uL-antifluid (rabbit anti mouse Caspase-3 multiclonal antibody (1:150), stay overnight at 4℃; add goat anti rabbit IgG at 37℃for 20min; add SABC at 37℃for 20min; every step washed by PBS for 3min 3 times; DAB developer, slightly restain by hematoxylin, dehydrate, transparent and mount. Use the microgram analysis system to collect images, and analyze spectrodensity of positive cells.5. Measuring TNF-aprotein by immunohistochemistryUse SABC to measure positive cells which express TNF-aprotein. Add the slice with 50uL-antifluid(rabbit anti mouse TNF-amulticlonal antibody (1:200)), stay overnight at 4℃; add goat anti rabbit IgG, at 37℃for 20 min; add SABC at 37℃fro20min; every step washed by PBS for 3min 3 times; DAB developer, slightly restain by hematoxylin, dehydrate, transparent and mount. Use the microgram analysis system to collect images, and analyze spectrodensity of positive cells.Statistical analysisAll datas were demonstrated by mean±standard deviation(x±SD), using SPSS 16.0 and Excel software for data processing and analysis of variance(ANOVA), comparing each two groups when (?)atistically significant and using spearman correlation analysis, P<0.05 when difference significant.Results1. Measuring brain water contentAfter ICH,the brain tissue water content began to increase at 6h,3d reached the peak,7d gradually reduced, as compared with the control group, but there are significant differences (P<0.05).2. Measuring EB content of the brain tissueExperimental ICH in rats brain tissue EB content began to increased after ICH 6h, 3d reached the peak,7d group was gradually reduced, as compared with the control group, but there are significant differences (P<0.05).3.Expression of Caspase-3 after cerebral hemorrhage:After ICH 6h, there was a weakly positive expression of Caspase-3 surrounding the hematoma and in cerebral cortex of the same side, mainly expressed in kytoplasm. Atfer 1d there were a great quantity of positive neurons began to emerge,3d reached peak with the deepest coloration in nucleus, which meant there was nuclear transfer after schizolysis of capases-3. The quantity decreased after 7d(P<0.05).4.Expression of NF-κB after cerebral hemorrhageNF-κB positive cells surrounding the hematoma began to expessed at 6h, continued to increase after 1d, reached peak at 3d and after this began to decrease, which was higher for continuous 7d than control(P<0.05). It was expressed mainly in gliocytes and some neurons, it was positive when nucleus was cinnamomeous and cannot be stained by hematoxylin. NF-κB was also expressed on the opposite side of hematoma. In control group, we found extremely few positive cells.5.Brain hemorrhage the expression of TNF-α:After ICH 6h, TNF-abegan to express in rats hematoma surrounding tissue,3d reached the peak, the group and there exist differences between the control group (P <0.05).The results show that the brain tissue around the hematoma immunohistochemical increase the number of positive cells, showing the dynamic change trend, observed under the microscope with the optical nerve cells in interstitial edema and surrounding tissue edema, inflammatory cell infiltration consistent with the dynamic change trend.DiscussionWe injected autoblood from femoral artery into right caudate nucleus to make models of cerebral hemorrhage, used immunohistochemistry to measure NF-κB、Csapase-3、TNF-αand found out that there was a change of expression of both NF-κB、Csapase-3、TNF-αand protein after cerebral hemorrhage. Recently, many researches confirmed that apoptosis was involved in secondary lesion after cerebral hemorrhage. Many researches have also confirmed that this inflammation exists surrounding hematoma, which is thought to result in apoptosis after cerebral hemorrhage. Time of inflammatory cell infiltration is positive correlant with time of apoptosis. In this experiment the application of adjacent sections separately Caspase-3, TNF-a and NF-κB immunohistochemistry, three-positive cells are basically the same scope and time-varying. Tip Caspase-3 in neuronal apoptosis after cerebral hemorrhage plays an important role in regulation. NF-κB signaling pathway is the intracellular signal transduction pathway downstream of the most important pathway involved in inflammatory response and apoptosis in the pathological and other important physiological processes. This experiment was found, NF-κB, Caspase-3 expression and nuclear positive rate and the increase of TNF-a were positively correlated, suggesting that an increase in NF-κB expression in nerve cells accompanied by activation of Caspase-3, that is, Caspase-3 nuclear translocation. An extensive literature has confirmed the existence around the hematoma after intracerebral hemorrhage in the above-mentioned reaction, and that it could lead to brain hemorrhage cell apoptosis. Infer from this that NF-κB imediated Caspase-3 signaling pathways involved in brain inflammatory response after signing out of the blood and increased neuronal damage and apoptosis.Conclusion1. Caspase-3, TNF-a and NF-κB in rat brain hemorrhage after the increased expression of brain tissue around the hematoma.2. NF-κB imediated Caspase-3 signaling pathways involved in brain inflammatory response after signing out of the blood and increased neuronal damage and apoptosis.更多还原