【作者】 许愿忠；

【导师】 曾志成；

【作者基本信息】 中南大学 ， 神经生物学， 2007， 博士

【摘要】 正常脑衰老过程中其免疫反应能力常发生改变，其中一个重要的指标是促炎性细胞因子水平明显升高。正常老年脑内的胶质细胞在促炎性细胞因子刺激时活化明显增强，其免疫应答反应是否同样存在这种年龄相关性增强尚不清楚。升高的促炎性细胞因子水平参与了机体的睡眠调节，而衰老因素可以影响该过程；衰老过程中睡眠丢失可否导致与脑老化过程相似的脑内免疫反应改变?其是否由促炎症细胞因子表达水平改变所致?尚未见报道。目的通过观察促炎性细胞因子刺激在不同年龄小鼠脑内诱导的免疫应答反应，以及快眼—运动（REM）睡眠剥夺对成年大鼠脑内胶质细胞活化、炎症细胞浸润和促炎性细胞因子表达水平的影响，探讨促炎性细胞因子在正常脑衰老过程中诱导免疫应答反应和参与睡眠调节过程的可能作用。方法利用实时定量逆转录聚合酶链反应（real time RT-PCR）技术，分别观察两种主要的促炎症介质TNF-α和IFN-γ侧脑室注射4小时后对青年（3～4月）和老年（21～23月）C57BL／6J小鼠脑内炎症反应相关分子ICAM-1、MIG、MMP-12、SOCS-1和SOCS-3基因转录水平的影响，PBS注射动物作为对照；并检测未经细胞因子处理组动物脑内TNF-α受体基因TNFR2的转录水平改变。基于早期基因转录水平改变，TNF-α侧脑室注射48小时后，检测青（3～4月）、中（8～9月）和老年（19～21月）C57BL／6J小鼠脑内血-脑屏障通透性相关重要细胞粘附分子ICAM-1蛋白表达和3种重要T淋巴细胞亚型的浸润情况，PBS注射动物作为对照；在成年SD大鼠，REM睡眠剥夺60小时后运用免疫组化、免疫荧光和荧光双标技术分别检测其脑内胶质细胞活化、炎症细胞浸润和促炎性细胞因子TNF-α表达水平的改变，应激对照组和正常动物作为对照。结果基因转录水平测定显示：在老年小鼠脑内，TNF-α刺激诱导明显增强的ICMA-1基因转录（P＜0.05），而IFN-γ刺激诱导明显增强的MMP-12、SOCS-1和SOCS-3基因转录（P＜0.05）；TNFR2基因转录水平在老年对照组小鼠脑内亦明显增高（P＜0.05）。免疫组化和Western blotting结果均显示TNF-α刺激诱导的ICMA-1蛋白表达在老年组动物明显增强（P＜0.05），荧光双标结果显示活化的星形胶质细胞和小胶质细胞均表达ICAM-1。与青、中年组动物相比，TNF-α刺激诱导的浸润CD³⁺、CD⁴⁺和CD⁸⁺T淋巴细胞平均数目在老年组小鼠脑内明显增加（P＜0.05）。与应激对照和正常组动物相比，REM睡眠剥夺诱导大鼠脑内星形胶质细胞和小胶质细胞活化明显增强，MHC-Ⅱ类抗原表达和ED-2阳性巨噬细胞浸润明显增加，并伴有TNF-α表达水平明显上调（P＜0.0001）；双标结果显示活化的星形胶质细胞而非小胶质细胞表达TNF-α上调。结论1．促炎性细胞因子可以放大和延长其在衰老脑内的促炎症反应功能；2．正常衰老脑对促炎性细胞因子刺激产生免疫应答反应增强、易感性升高；3．衰老个体睡眠行为改变可以影响脑内胶质细胞活化和炎性反应，促炎性细胞因子在此过程中发挥着重要的作用；4．促炎性细胞因子、衰老脑炎症反应与衰老个体睡眠节律改变三者之间存在着非常密切的联系，相互影响。更多还原

【Abstract】 Normal brain aging accompanies alterations in inflammatoryactivities, and one vital this alteration is that increased production ofpro-inflammatory cytokines. It has been demonstrated that activation ofglial cells in the brain responsing to an acute pro-inflammatory cytokineschallenge is enhanced at an advanced age. So far, it remains largelyunknown whether or not there is an age-related change inimmune-associated responses in normal aging brain to an inflammatorychallenge. The change that increased level of pro-inflammatory cytokinesalso contributes to sleep regulation, and this process is impacted bynormal aging. However, little information is available on whether or notsleep loss with aging can elicit same changes in immune-associatedresponses that happen in normal aging brain, as well as this changedepend or not on circulating of pro-inflammatory cytokines level.Objective To investigate the possible roles of pro-inflammatorycytokine in immune-associated responses in normal aging brain, as wellas in sleep regulation during normal aging. For this purpose, theimmune-associated responses in the mouse brain of different ages to anacute pro-inflammatory cytokines challenge was observed, the activationof glial cells, the recruitment of inflammatory cells, and the expressionlevel of pro-inflammatory cytokines in adult rat brain following rapideye-movement (REM) sleep deprivation was also studied.Methods Quantitative real time RT-PCR was employed to determineearly transcriptional changes of genes involved in the response to acuteicv administration of the cytokines tumor necrosis factor (TNF)-αorinterferon (IFN)-λin young (3-4 months) and old (21-23 months) mice.Control experiments were performed in parallel with icv injections ofsaline. After obtaining information on such early response, the expressionof the protein intracellular adhesion molecule (ICAM)-1, which plays akey role in the regulation of blood-brain barrier permeability, wasexamined with Western blotting and immunohistochemistry at 48 h afterTNF-αor saline injections in mice of different ages (3-4 months, 8-9 months and 21-23 months). The recruitment of CD3~+, CD4~+ and CD8~+Tcells in the brain was examined in the same paradigm withimmunohistochemistry. Finally, glial cells and macrophages, majorhistocompatibility complex antigen classⅡ(MHC-Ⅱ) and TNF-αwerestudied in adult rats following 60 h of sleep deprivation.Immunohistochemistry (in bright-field and in fluorescence microscopy,including double immunofluorescence) was used in this part of theinvestigation.Results Real time RT-PCR revealed that the up-regulation elicited byTNF-αon the expression of the ICAM-1 transcript, and by IFN-γon theexpression of MMP-12, SOCS-1 and SOCS-3 transcripts was significantlyincreased in the brain of old mice compared to young ones (P＜0.05). Inaddition, the expression of the TNF receptor TNFR2 was significantlyenhanced in the septum and hippocampus of both cytokine-injected andcontrol (saline-injected) old animals (P＜0.05). At 48 h after TNF-αicvinjections, immunohistochemistry and Western blotting revealed also asignificant up-regulation of the expression of the ICAM-1 protein in thebrain of old mice as compared to younger animal groups (P＜0.05).Double immunofluorescence revealed that ICAM-1 expression in bothastrocytes and microglia.The study of T cell recruitment in the brain pointed out the interestingfinding of enhanced recruitment of lymphocytes in the brain of old micein response to cytokine administration (P＜0.05)In adult rats, after 60 h of sleep deprivation, the activation of bothastrocytes and microglia, the expression of MHC-Ⅱantigen and of TNF-α,the number of activated macrophages were all significantly enhancedcompared with control subjects (P＜0.0001). Double immunostaining ofTNF-αcombined with glial antigens (the astrocyte marker glial fibrillaryacidic protein or the microglial marker OX-42) showed that astrocytesexpressed TNF-αin this paradigm.Conclusionⅰ) The pro-inflammatory effects of pro-inflammatorycytokines are amplified and prolonged in normal aging brain;ⅱ) Animmune hyperresponsiveness and injury susceptibility topro-inflammatory challenge is elicited in normal aging brain,ⅲ) Variation in sleep rhythm exerts impacts on the activation of glial cells and theresponsiveness of inflammatory-associated response during aging, andpro-inflammatory cytokines play an important role in this process,ⅳ)Altogether these results reveal that pro-inflammatory cytokines areclosely correlated with immune-associated responses in normal agingbrain and involved in sleep regulation of aged individuals.更多还原