【作者】 赵逸松；

【导师】 薛一雪；

【作者基本信息】 中国医科大学 ， 生理学， 2004， 硕士

【摘要】 前言 血脑屏障（blood brain barrier，BBB）由脑毛细血管内皮细胞及细胞间紧密连接、基膜、周细胞、星形胶质细胞脚板和极狭小的细胞外隙构成。BBB限制药物向脑组织的充分转运，因此限制了脑部疾病的治疗。多年来科学家们一直在试图寻找一个安全高效的方法增加药物向脑组织转运以提高脑部疾病的治疗疗效。其中最引人注目的是利用血管活性物质增加毛细血管通透性的特性选择性开放BBB。正常脑组织的毛细血管对血管活性物质增加其通透性的作用具有抵抗性，但是脑肿瘤组织的毛细血管或损伤部位的脑组织毛细血管丧失了抵抗血管活性物质增加其通透性的能力。 神经胶质瘤在脑肿瘤中最常见，而且由于其易复发，治疗效果较差。近年来许多研究者利用神经胶质瘤动物模型进行血管活性物质开放血肿瘤屏障（blood-tumor barrier，BTB）的研究工作。多项研究已经证实血管活性物质引起BTB开放是有选择性的，即仅增加脑肿瘤组织毛细血管通透性而不影响正常脑组织毛细血管通透性，大大减小了药物对于正常脑组织的细胞毒性效应。目前最有效的且相对安全可控的选择性开放BTB的血管活性物质是缓激肽或其类似物RMP-7（receptor-mediated permeabilizer-7，RMP-7）。研究证实缓激肽增加BBB通透性效应是通过缓激肽B₂受体所介导的。在临床实验中发现缓激肽或其类似物RMP-7引起的BTB通透性增加的差异非常大。这种差异的存在限制了缓激肽及其类似物RMP-7在临床上的推广和应用。我们推测这种差异的存在可能与脑肿瘤组织的缓激肽B₂受体表达水平有关。认清这种差异的原因是目前急待解决的问题。 本研究采用神经胶质瘤患者术后肿瘤组织标本，测定神经胶质瘤组织的缓激肽B₂受体的表达水平，与其病理分级相比较，分析神经胶质瘤缓激肽B₂受体表达水平与其病理分级的关系，试图从理论上弄清缓激肽及其类似物RMP-7引起的BTB通透性增加存在显著差异的原因，从而在临床治疗中，筛选出适合应用缓激肽及其类似物选择性开放BTB转运药物进行治疗的神经胶质瘤患者，切实提高神经胶质瘤的疗效和神经胶质瘤患者的生存质量。材料和方法 一、实验材料 1.标本本研究收集中国医科大学第一临床学院神经外科2002年5月一2003年4月间手术切除的神经胶质瘤标本28例。其中男性16例，女性12例，年龄5一75岁，平均年龄43.58岁。选取肿瘤边缘正常脑组织2例作对照。病理分级标准采用20(刃年世界卫生组织（wnrldh已dth org肛川a-tion，wHO）中枢神经系统肿瘤分类标准。 2.主要试剂羊抗人缓激肤B:;受体抗体和ECL（ enhanced chanl吐扭ni-nescence，ECL）试剂盒均购于美国SantaC二Biotechnology公司，辣根过氧化物酶标记的兔抗羊免疫球蛋白和SP（streptavi击n一pero石dase，SP）免疫组化试剂盒均购于北京中山公司。 3.主要仪器匀浆机;台式低温超速离心机;电泳仪;转印仪;恒冷冰冻切片机;Luzex一F实时图像分析系统。， 二、实验方法 x .H&E（hematoxylin and eosin，11&E）染色进行脑肿瘤的病理诊断及分级。 2.根据H&E染色结果从标本中筛选出神经胶质瘤I级8例，神经胶质瘤n级8例，神经胶质瘤m级8例，每个病理级别为一组，共三组。 3 .Westem Blot法测定缓激肤B:受体的表达水平，分析神经胶质瘤BZ受体表达水平与其病理分级的关系。， 4.免疫组化sP法测定缓激肤B:受体的表达水平，分析神经胶质瘤BZ受体表达水平与其病理分级的关系。， 5.图像分析 Westem Blot结果用Cha面玩鳍er5500 V2.仍软件扫描，用凡uor Chen2.0软件进行定量分析测得IDV。免疫组化结果用L山妃x一F实时图像分析系统采集照片并进行半定量分析测得目标总面积占选区面积百分比值。 6.统计分析 采用SpSS n.0统计分析软件对实验数据进行统计分析，结果用平均值土标准差表示。统计分析方法采用独立样本t检验，P<0 .05有统计学意义。采用相关分析和一元线性回归对神经胶质瘤B:受体表达水平与其病理分级进行相关性分析。实验结果 一、H&E染色进行脑肿瘤的病理诊断及分级 28例病例中有26例为神经胶质瘤，1例为脑膜瘤，1例为脑脓肿。26例神经胶质瘤中I级为9例，n级为9例，m级为8例，IV级为0例。‘ 二、w‘tem Blot法侧定缓激肤BZ:受体的表达水平 正常脑组织不表达B:受体，三组神经胶质瘤组织B:受体表达水平存在差异。测得的B:受体均为双蛋白带，77 kDa为B:受体，低分子量蛋白带为B:受体的衍生物。每个样品均进行双蛋白带侧定。由于正常脑组织标本只有两例，所以未作正常对照组和各组间对比。三组间B:受体表达水平取IDV两两相比，I级与n级（3华比0.88土5119.%对51354.25士6168.77，n=8，P<0.05），I级与111级（3夕铭0.88土5119.96对73032.13士8802.71，n=8，P<0.001），11级与111级（51354.25土6168.77对73032.13土8802.71，n=8，P<0.001），组间差别均有统计学意义。三组神经胶质瘤组织B:受体表达水平为I级<n级<m级。神经胶质瘤B:受体表达水平与其病理分级呈显著正相关，直线回归方程为y二1幻75 .62x+210更多还原

【Abstract】 ObjectiveBlood-brain barrier ( BBB) is composed of capillary endothelial cells, intercellular tight junction, base membrane, astroglial processes. BBB limits adequate delivery of drugs to brain. Scientists have been looking for a safety method to open the BBB. Now the most noticeable method is opening BBB by vasoactive compounds. Vasoactive compounds can increase the permeability of blood vessel. Normal brain capillaries appear to resist the efforts of vasoactive compounds. In contrast, brain tumor capillaries or injured brain capillaries appear to lose the ability to resist the effects of increased permeability by these vasoactive compounds.Malignant brain tumors represent a serious medical challenge for they are the second leading neurological cause of death, with the majority of patients dying within a year of initial diagnosis. Brain tumor capillaries have a blood -tumor barrier ( BTB) that limits adequate delivery of antitumor agents to tumor tissue. Gliomas are the most prevalent type of brain tumor. Recently, biochemical opening of the BTB with vasoactive compounds have been used to increase drug delivery to gliomas. Studies have demonstrated that this approach do not increase drug delivery in normal brain, but result in a selective, significant increase in permeability in the tumor. Currently, the most effective compound to selectively open the BTB is bradykinin or its analog, receptor-mediated perme-abilizer-7 ( RMP-7). The selective increases in BTB permeability induced by bradykinin are mediated solely by B2 receptor. In clinical study there is very great difference of increase of BTB permeability induced by bradykinin in brain tumors with the same diagnose. The variability in response of tumors to bradykinin is likely related to the expression level of bradykinin B2 receptor of tumor. It is the urgent task to recognize the reason of the variability in response of braintumors to bradykinin.This study used fresh glioma samples to determine expression level of bradykinin B2 receptor of glioma with different pathological grade and analyzed the difference of bradykinin B2 receptor expression level among gliomas with different pathological grade. The result would supply experimental basis for clinical application of bradykinin.Materials1. Experimental specimens: fresh tissue samples were obtained from 28 patients with brain tumor who underwent surgery in the Department of Neurosurger-y, the Affiliated Hospital of China Medical University, Shenyang, China. After resection the samples were immediately frozen in liquid nitrogen then kept at -70 C. Two cases of normal brain tissues of the edge of tumor were selected as control.2. Experimental reagents: goat anti human bradykinin B2 receptor antibody and enhanced chemiluminescence kit ( Santa Cruz Biotechnology, U. S. A. ) ; streptavidin-peroxidase (SP) kit (Zhorigshan Company, Beijing).3. Experimental instruments: stir apparatus, low temperature centrifugal machine, electrophoresis apparatus, transmark apparatus, freeze microtome, Luzex-F image analysis system.MethodsThe clinicopathologic findings were diagnosed by reviewing all hematoxylin and eosin (H&E) stained tissue sections. The tumor grade was determined according to the 2000 World Health Organization (WHO) Central Nervous System Tumor Classification.According to the result of H&E screened 8 cases of grade 1, 8 cases of grade II, 8 cases of grade III. Devide the specimens into 3 groups. Every grade was as one group.To determine the bradykinin B2 receptor expression level of glioma, Immu-nohistochemistry and Western blot methods were used.Integrated Density Value (IDV) for the B2 receptor protein doublet band was calculated by Fluor Chen 2. 0 software from scanned immunoblot by Chemi Imager 5500 V2.03 software. The distribution of cells immunopositive for anti-B2 receptor antibody was analysed semiquantitatively by Luzex-F Real-Time Image Analysis System. Target area/election district area ratio was calculated.Data from Immunohistochemical and We更多还原