【作者】 李玲；

【导师】 水泉祥；

【作者基本信息】 浙江大学 ， 儿科学， 2003， 博士

【摘要】 脑源性神经营养因子(brain derived neurotrophic factor，BDNF)是神经营养素第二个家族成员，广泛分布于中枢神经系统，与其最适配体TrkB受体结合激活酪氨酸激酶(PTK)，从而激发细胞内各种信号传导，发挥生物学效应。BDNF在中枢神经系统发育过程中，对各种神经元的生存、分化、生长起着重要作用，也是维持成熟的中枢神经系统神经元的生存及执行其正常生理功能所必需的，并促进损伤后的再生。国内外对BDNF及其受体的结构、信号传导、分子克隆和生理条件下基因表达与调控以及BDNF在神经系统的生物学作用进行了大量的研究，近年来在低血糖、脑缺血缺氧和脑创伤以及伤害性刺激的情况下，研究BDNF及其受体的表达和作用，尤其是BDNF对视、听神经细胞保护及损伤后再生的作用有所研究，旨在开发BDNF用于脑损伤和视、听障碍的治疗。大量实验显示脑缺血缺氧后，脑内广泛区域出现BDNF及其受体mRNA表达明显增加，外源性BDNF治疗缺血缺氧所致脑损伤可挽救脑神经元的存活，恢复记忆功能，尤其是对发育早期缺血缺氧脑损害的作用更突出。在耳蜗内应用外源性BDNF治疗耳聋，能提高和保护听神经细咆存活并诱导其再生。最近报道，在体外培养中，经抗原刺激后免疫细胞能分泌BDNF，并具有神经保护活性，而在散发性脑白质病变和多发性硬化患者的脑组织中也发现渗出的炎症细胞有高表达的BDNF。关于BDNF及其受 浙江大学博士学位论文 体在细菌感染等炎症组织中的表达和作用国内外尚未见报道。尽管有报道在生理状态下糖皮 质檄素能刺激皮质和海马中BDNFmRNA的表达，但在不同脑损伤状态下怎样表达尚不清楚。 细菌性脑膜炎是儿科常见的中枢神经感染性疚病，尽管新的、强有力的抗生素不断问世和应 用以及支持性医疗监护质量的提高，使死亡率有所卜降，但存活者神经系纷后遗症的发十率 仍然不变。细曲进入中枢神经系统后，它们的繁殖触发了宿主复杂的反应，可使血脑屏障破 坏以及整体或局部缺血的脑血管炎性改变，最终导致功能性和结构性的脑损伤（癫痈、智力 低下和肢体瘫痪），炎性病变常波及颅神经，受损严重临床上常出现耳聋、大明和面神经麻 痹等后遗症。我们推测在细曲性脑膜炎时可能有BDNT及其受体表达增加，外源性BDNF可能 兑有神经保护作用。 第一部分BDNF及其受体在实验性细菌性脑膜炎表达动态变化的研究 目的 研究细曲性脑膜炎 BDNF、BDNF mRNA和 TrkB。RNA在脑织织内表达的变化特点以及抗生 素和地塞米松（DEX）治疗后表达的变化，探讨细菌性脑膜炎后遗症高发的可能机制和 BDNF 在细曲性脑膜炎的可能作用。 方法 采用肺炎链球菌＊ 型菌株和等量的生理盐水脑池山注射，建立 3周龄大鼠细菌性脑 膜炎模刑和正常对照模刑，用免疫纽化染色和原位杂交技术，分别于感染后24h、48h、5 天，以及感染24h经抗生素和DEX治疗后，检测BDNF、BDNF mRNA和TrkB mRNA在脑组织内 表达， 结果 2 浙江大学博士学位论文 一、BDNFmRNA的表达 二、感染组 细菌感染后 24h，BDNF mRNA ／f脑组织）“泛部位有明显表达，大脑皮层和海 马均较突出，m性杂交信号与对照组相比，有显著性差异（HO．of）。48h后表达逐渐－卜降， 与 24h相比有显著性差异（HO．01），至第 5天 BDNF。RNA表达仍高丁对照织（p＜0．05）。对 照组注射后 24h、48h和 5大 BDNF mRNA无显著性芦异（KO．05）。 2、抗生素组抗生素治疗后 BDNF mRNA在大脑皮层和海马）”泛部位表达均很低，多数区 域无m性杂交信号，与感染后24h相比有显著性差异（WO．01）。与感染后和对照组第5天 相比，也均有显著性差异（HO．of）。 3、抗生素加 DEX组在抗生素加 DEX治疗后 BDNF mRNA在脑组织有明显表达，皮层、 海马均有较强的杂交信号，与抗生素组相比有而著件芙异（WO．of），但仍低于感染后24h 组，差异有显著性意义（AO．of）。与感染后 5天比较，无显著性差异（HO．05），而高于对 照纲 5天水平，有品关件芙异（KO．of）。 4、在软脑膜、蛛网膜下腔、脑室内，渗出的炎性细胞可见BDNF mRNAgn性杂交信号， 对照绢未见炎件掺出。 二、BDNF蛋白的表达 1、感染织细菌感染后24h，大脑皮层和海马广泛区域80抑蛋白表达明穴增加，与对 照纽相比，差异有显舍意义（HO．of）。4Sh后逐渐下降，与24h相比差异有显著意义更多还原

【Abstract】 Brain derived neurotrophic factor (BDNF), which is the second member of neurotrophin family and widely distributed throughout the central nervous system, activates various signal transduction pathways in cells and exerts its biological function by binding with the high-affinity TrkB receptor to activate Protein Tyrosine Kinase. BDNF not only plays an important role in regulating neuronal survival, differentiation, proliferation, maturation and outgrowth during the development of the central nervous system (CNS), but also is essential in retaining the survival and normal physiologic function of mature neurons. There have been a large number of results of investigations on the structure, signal transduction and molecular cloning of BDNF and its receptor, TrkB, as well as the expression and control of BDNF gene under physiologic situation and its biologic role in nervous system. In order to develop the application of BDNF in the treatment of brain damage and optical and auditory impediment, recently, the expressions and effects of BDNF and TrkB have been investigated under the situation ofhypoglyceraia, ischemia, traumatic brain damage and noxious stimulation, in particular, the effects of BDNF on protecting and regrowing optical and auditory neurons after some injury have been studied. A lot of experiments have shown that the expression of BDNF and its receptor in extensive region of brain is obviously elevated in response to ischemic and hypoxic brain damages, the administration of exogenous BDNF can increase the population of survival brain neurons and recover memory function after ischemic and hypoxic brain damage, especially, it has more potent neuroprotective action on in ischemic-hypoxic brain injury in the developing brain. Some studies have shown that BDNF can enhance and protect survival of spiral ganglion cells and induce them regrowth, when exogenous BDNF was applied in cochlea after deafness.Latest, reports have released that human immune cells activated by specific antigen in vitro can secrete BDNF which can support neuronal survival, BDNF immunoreactivity is demonstrable in inflammatory infiltrates in the brain of patients with acute disseminated leukoencephalopathy or multiple sclerosis. The expression and effects of BDNF and TrkB in the inflammatory tissue infected by bacteria, however, aren’ t clear. Although it has been reported that glucocorticoid can stimulate the expression of BDNF in cerebral cortex and hippocampus under normal physiology situation, the expression of BDNF in various brain injury is still unclear.Bacterial meningitis is the most common serious infection of the central nervous system. Though newer and more potent antibiotics are continually developed and applied, and there are more and more advanced nurse, the mortality and permanent neurologic deficits still high occur following bacterial meningitis inchildren. Once bacteria have gained access to the central nervous system, their multiplication triggers a complex host response. Alteration of the cerebral vasculature, with disruption of the blood brain barrier and global and focal ischemia, ultimately lead to functional and structural brain damage. Inflammation also often affect cochlear inner hair cells and cranial nerve, which leads to many sequelaes involving in deafness, blindness, facial paralysis and retardation in serious patients. It is essential to emphasize possible therapeutic strategies to prevent its harmful consequences. We infer that the expressions of BDNF, BDNF mRNA and TrkB mRNA in CNS may enhance in bacterial meningitis, and exogenous BDNF may have neuroprotective effect in bacterial meningitis.Part I The dynamic change of expressions of BDNF and its receptor in experimental bacterial meningitisObjectiveTo investigate the expressions of BDNF, BDNF mRNA and TrkB mRNA in brain after bacterial meningitis and their changes after antibiotic and dexamethasone used, in order to discuss the possible mechanisms of mortality and high occuring neurologic deficits following bacterial meningi更多还原