【作者】 朱威；

【导师】 李正莉；

【作者基本信息】 华中科技大学 ， 人体解剖学与组织胚胎学， 2010， 博士

【摘要】 小胶质细胞(MG)约占胶质细胞总数的20%,属巨噬细胞系,是中枢神经系统病理变化的传感器,在中枢神经系统的损伤,炎症、癫痫等病理过程中均有MG持续活化、增殖现象。MG活化后分泌大量细胞因子和活性分子,包括IL1β, TNFα和NO等,这些过量分泌的物质对神经元具有兴奋毒性损伤作用,与中枢神经系统神经变性疾病(如癫痫)所致神经元的丢失有关。因此活化的MG被普遍认为是一种细胞毒性效应细胞。TNFα是一种重要的炎性因子,在脑内神经受损部位,其主要来源于活化的MG。研究表明,在高表达TNFα的转基因小鼠具有年龄依赖性神经变性改变和自发性癫痫；临床使用高剂量的TNFα进行抗肿瘤治疗会诱发癫痫。而使用单克隆抗体拮抗中枢TNFα或抑制小胶质细胞激活,均能有效降低神经元对癫痫的易感性,有助于提高其存活率。虽然上述资料提供的证据表明MG活化状态及脑内高水平的TNFα可能与癫痫所致神经元损伤有一定联系,但是,很少有详细的实验资料证明,被活化的MG所分泌的内源性TNFα在海马神经元的兴奋毒性事件中具有重要作用。因此,本研究以致痫剂海人酸(kainic acid, KA)激活MG,以美满霉素(minocycline, MC)作为抑制MG活化的工具药,通过在体和离体实验探讨上述问题。本研究共分三个部分：第一部分KA对MG的激活作用及促TNFα分泌效应研究为了证实KA对大鼠MG具有激活作用及促进TNFα分泌的效应。本实验先分离纯化培养新生大鼠大脑皮质MG,以100μM KA激活MG或以MC预处理抑制MG的活化,然后应用OX-42免疫染色观察经上述不同处理后MG的形态学变化；分别采用RT-PCR和Western blot法检测MG内TNFαmRNA和蛋白的表达情况；采用Elisα法检测MG条件培养液内TNFα的含量。OX-42免疫组化结果显示,KA作用MG 2hr后,MG胞体增大、突起回缩呈阿米巴状,OX-42的免疫反应较正常组和MC+KA组明显增强。结果显示,MG内TNFαmRNA和蛋白的含量在KA组明显高于正常组；而在MC+KA组则显著低于KA组。Elisα检测结果显示,MG条件培养液中TNFα的含量在KA组明显高于正常组；而MC+KA组则明显低于KA组。结果提示,KA对MG有显著的激活作用,活化的MG合成、分泌TNFα显著增多。MC预处理能抑制MG的活化进而拮抗KA对MG的上述效应。第二部分KA活化的MG条件培养液对海马神经元兴奋性的影响及TNFα抗体的拮抗效应海马神经元因具有自发放电,兴奋阈值低等特征而与癫痫发病关系密切。中枢神经病变时脑内表达的各种炎性细胞因子能显著影响海马神经元兴奋状态,其机制可能与激活神经元上的相应受体和离子通道有关。在第一部分的实验中,我们已经证实活化的MG合成分泌TNFα显著增多。为了进一步探讨KA活化的MG条件培养液(KA-MCM)对海马神经元的兴奋作用及KA-MCM内的TNFα在这一事件中的重要作用。本实验通过离体和在体实验,采用膜片钳和诱发电位电生理技术、RT-PCR和Western blot方法,研究KA-MCM对海马神经元钙电流、群峰电位、相关兴奋性蛋白NMDAR1、iNOS的影响,并观察TNFα抗体加入KA-MCM后所产生的中和拮抗效应。膜片钳及群峰电位记录表明,KA-MCM能显著增加海马神经元钙电流密度；增强在体大鼠海马CA3区群峰电位。RT-PCR和Western blot检测表明KA-MCM显著增加海马神经元和海马组织NMDAR1和iNOS的mRNA及蛋白质表达,而预先在KA-MCM中加入TNFα抗体则上述效应明显减弱。结果表明,活化的MG来源的TNFα对在体及离体的海马神经元具有极强的神经兴奋作用,由此提示它可能增加机体对癫痫发作及神经元损伤的易感性。第三部分KA活化的MG来源的TNFα对海马神经元的促凋亡作用。研究表明：在颞叶癫痫患者以及电点燃或化学点燃所致的各种癫痫研究模型中,脑内的病理改变有神经元丧失和与胶质细胞增生,且神经元丧失以神经元凋亡为主要方式。在前二部分实验的基础上,我们进一步探讨KA活化的小胶质细胞培养液(KA-MCM)对海马神经元的促凋亡作用以及TNFα抗体的拮抗效应。本实验通过离体和在体实验,采用原位末端标记(TUNEL)检测,免疫组化、RT-PCR方法,观察KA活化的MG条件培养液(KA-MCM)及TNFα抗体加入KA-MCM后(anti-TNFa-KA-MCM)对海马神经元TUNEL阳性细胞,凋亡关键酶caspase-3 mRNA及其蛋白表达的影响。TUNEL检测结果显示,经UT-MCM、KA-MCM、anti-TNFα-KA-MCM三种不同MCM孵育的海马神经元,在24,48小时时间段其TUNEL阳性细胞数稀少,组间无显著性差别,但在72小时KA-MCM组凋亡细胞显著增多,anti-TNFα-KA-MCM组凋亡细胞数明显少于KA-MCM组。RT-PCR结果显示,大鼠侧脑室注射KA-MCM48小时海马caspase-3 mRNA表达有所增加,72小时明显增加,相同时间点anti-TNFa-KA-MCM组caspase-3 mRNA表达较KA-MCM组明显减弱。caspase-3免疫组化结果与上述RT-PCR结果趋势相似。结果表明,活化的MG来源的TNFα对在体及离体海马神经元具有明显的促凋亡作用,可通过激活caspase-3参与介导该过程。小结：KA活化的MG高表达和分泌TNFαKA-MCM可显著增强大鼠在体海马群峰电位；增加离体海马神经元钙电流密度以及海马神经元或海马组织NMDAR1,iNOS和caspase-3 mRNA和蛋白表达；经KA-MCM孵育的海马神经元凋亡细胞显著增多。而若在KA-MCM中预先加入抗TNFα抗体,可明显抑制上述效应。本研究提供的上述结果表明,KA活化的MG来源的TNF-α在离体和在体的情况下,具有神经兴奋毒性作用,可诱导海马神经元凋亡发生。根据本实验结果,我们认为TNFα对海马神经元的兴奋毒性作用可能与激活神经元谷氨酸受体及电压门控钙通道,使胞内钙超载,一氧化氮(NO)过度生成、激活凋亡关键酶caspase-3等多种因素有关。神经免疫调节机制在癫痫的治疗研究领域是近年备受关注的热点,本研究为临床癫痫防治、合理开发神经保护药物提供了新的实验资料和理论依据。更多还原

【Abstract】 Microglia constitute 20% of the total glial cell population and belong to macrophages cell line. They are sensors for pathological events in the CNS. Sustained activation and proliferation of microglia were observed during the pathological progressions of injuries, inflammation and epilepsy in CNS.Activated microglia secret a variety of proinflammatory cytokine and toxic molecular, include IL1β, TNFa and NO et al. Those over secreted substance have excito-toxic effects on neurons and which are associated with neurpnal loss in neurodegeneration disease such as epilepsy. So activated MG are generally recognized as cytotoxic effecter cells.TNFa is an important inflammatory mediator which is mainly derived from MG at the site of injured neurons in the brain. Over expression of brain TNFa in transgenic mice is associated with the occurrence of age-dependent neurodegenerative changes and sporadic spontaneous seizures. It have been found that High dose of TNFa during anti-tumor therapy in clinic could induce seizures in human. However, antagonisms of TNFa using a monoclonal antibody or inhibition of MG activation decreases the seizure susceptibility of neurons and improves their survival rate..Although the evidence provided by above materials indicates that the activation state of MG and the high level of TNFa in the brain may have relationship with the seizure-induced neuronal injury, fewer detailed studies have demonstrated that the TNFa derived from activated microglia may play an important role in the event of excitotoxicity of hippocampal neurons. Therefore, in present study, KA was used as an activator and minocycline was used as a tool drug to inhibit the activation of microglia. This study is divided into three parts to explore the above matter through in vivo and in vitro experiments.To confirm that KA can activate rat’s MG and promote its secretion, MG from neonatal rat cerebral cortex were separated, purified and cultured.100μM KA was used to activate MG and minocycline pretreatment was used to inhibit the activation of MG. Then OX-42 immunostaining was performed to observing the morphological changes of MG undergoing above different treatments. RT-PCR and western blot was applied respectively to detecting the expression of TNFa mRNA and protein; Elisa was performed to testing the content of TNFa in microglial conditioned medium. The results of OX-42 immunohistochemistry showed that after being treated with KA for 2 hours, MG showed amoeba like shape with expanded cell bodies and retracted neurites, it’s OX-42 immunoreaction was obviously enhanced than those of the control group and the MC+KA group. The results of RT-PCR and western blot showed that the content of TNFa mRNA and protein in MG in KA group were obviously higher than those of the control group, and which was lower in MC+KA group than in KA group. The result of Elisa showed that TNFa levels in microglial conditioned media of KA group was significantly higher than those of the control group, whereas it was lower in MC+KA group than in KA group. The results indicate that KA significantly activated MG, and TNFa is over synthesized and secreted by activated MG. Pre-treatment with minocycline can inhibit MG activation and further antagonize above effects of KA.Because of the features of auto-discharge and low excitation threshold, hippocampal neurons have a close relationship with pathogenesis of epilepsy. A variety of cytokines expressed in the brain during the process of CNS diseases have an obvious effect on the excitability of hippocampal neurons, which may be rely on a mechanism of activation of their respective receptors and ion channels on neurons. In the first part of the study, we have confirmed that the synthesis and secretion of TNFa are increased in activated MG. To further explore the excitatory effects of KA-MCM on hippocampal neurons as well as the important role that KA-MCM contained TNFa plays in this event. Through in vitro and in vivo studies, we used patch clamp and evoked potential electrophysiological technology、RT-PCR and Western blot methods to study the effects of KA-MCM on hippocampal neuronal calcium current, population spike and the expression of related excitatory protein (NMDAR1 and iNOS). The antagonisms effects of TNFa antibody was observed when it was added into KA-MCM. The records of Patch clamp and population spike showed that KA-MCM significantly increased calcium current density in hippocampal neurons, and enhanced population spike on hippocampal CA3 region of rats in vivo. RT-PCR and Western blot showed that KA-MCM significantly enhanced the expression of mRNA and protein of NMDAR1 and iNOS in hippocampal neurons and tissues, whereas addition of anti-TNFa to KA-MCM significantly eliminated such effects. The results indicated that activated MG derived TNFa have strong excitatory effects on hippocampal neurons in vivo and in vitro and which can increase the body susceptibility to seizure and neuronal injury.Studies showed that pathological changes of brain include neuron loss and gliocyte hyperplasy in chemical or electrical kindling model of epilepsy as well as patients with temporal lobe epilepsy, and apoptosis is the major way of neuronal loss. Based on above two part of the studies, here we further explore the pro-apoptosis effects of KA-MCM in hippocampal neurons and the antagonistic effects of anti-TNFa. Through in vitro and in vivo studies, by using TUNEL detection, immunohistochemistry and RT-PCR method, we observe the effects of KA-MCM and anti-TNFα-KA-MCM (adding anti-TNFa into KA-MCM) on the expressions of TUNEL positive cells as well as caspase-3 mRNA and protein. The result of TUNEL detection showed that there are few TUNEL positive cells and no significant difference among them at 24,48 hr time period after incubated with three different kinds of MCMs, but they are obviously increased at 72 hr after incubated with KA-MCM. Apoptotic cells in anti-TNFa-KA-MCM group are less than those of KA-MCM group. The result of RT-PCR showed that the expression of caspase-3 mRNA is increased at 48 hr and increased obviously in 72 hr after intra-cerebroventricular injection of KA-MCM. At same time point they are less expressed in anti-TNFa-KA-MCM than in KA-MCM group. The result of caspase-3 immunohistochemistry has the same tendency with above result of RT-PCR. The results indicate that the TNFa derived from KA activated microglia can significantly promote apoptosis of hippocampal neurons. The activation of the caspase-3 is involved in this process.Summary:KA activated MG highly expressed and secreted TNFa. KA-MCM can significantly enhance hippocampal population spike in vivo, and increase calcium current、the expression of mRNA and protein of NMDAR1, iNOS、and caspase-3 of hippocampal neurons or hippocampal tissue in vitro. Apoptosis cells are increased significantly after being incubated with KA-MCM. However, the above effects of KA-MCM can be inhibited by adding anti-TNFa antibody into KA-MCM. The above results provided by present research indicate that TNFa derived from KA activated microglia is neuroexcitoxic to hippocampal neurons in vitro and in vivo, which can induce apoptosis on hippocampal neurons. On the basis of the results, We consider that the excito-toxic effect of TNFa on hippocampal neuron may be related to the activation of Glu receptors and voltage-gated calcium channels which result in calcium overload, nitric oxide (NO) production, and activation of caspase-3 (a key enzyme of apoptotic death), el al. The mechanism of neural-immune regulation is a hot spot of concern in therapy and study areas in recently years. This study provides new experimental materials and theory supports for the prevention and therapy of epilepsy, as well as the rational design of neuroprotective drugs.更多还原