促炎性细胞因子在老年啮齿类动物脑内诱导免疫应答增强及参与睡眠调节

发布时间：2018-08-08 17:07

【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CD~(3+)、CD~(4+)和CD~(8+)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。
[Abstract]:The immune response ability of normal brain is often changed during aging. One of the important indicators is a significant increase in the level of proinflammatory cytokines. The activation of glial cells in the brain of normal elderly is obviously enhanced when proinflammatory cytokines are stimulated, and the same age-related enhancement of its immune response is not clear. High levels of proinflammatory cytokines are involved in the body's sleep regulation, and the aging factor can affect the process. Can the loss of sleep in the process lead to changes in the brain immune response similar to the brain aging process? Is it the result of changes in the level of pro-inflammatory cytokines?
Objective to investigate the effect of rapid eye movement (REM) sleep deprivation on the activation of glial cells, infiltration of inflammatory cells and the expression of inflammatory cytokines in the brain of adult rats, and to explore the effect of proinflammatory cytokines on the aging process of normal brain by observing the immune response induced by pro-inflammatory cytokines in the brain of mice of different ages. The possible role of immune response and participation in sleep regulation.
Methods using real-time quantitative reverse transcriptase polymerase chain reaction (real time RT-PCR) technique, the transcriptional levels of ICAM-1, MIG, MMP-12, SOCS-1, and SOCS-3 genes in the brain of young (3~4 months) and C57BL / 6J mice were observed for 4 hours after the two major proinflammatory mediators, TNF- alpha and IFN- gamma lateral ventricle injection, respectively. Effects, PBS injected animals as control, and the changes in the transcriptional level of the TNF- alpha receptor gene TNFR2 in the brain of the untreated group. Based on the changes of the early gene transcription level, 48 hours after the TNF- alpha lateral ventricle injection, the blood brain barrier permeability in the brain of C57BL / 6J mice was detected in the middle (8~9 months) and the old year (19~21 months). Related important cell adhesion molecules ICAM-1 protein expression and 3 important T lymphocyte subtypes infiltration, PBS injected animals as control; in adult SD rats, REM sleep deprivation 60 hours after the use of immunohistochemistry, immunofluorescence and fluorescence double labeling technique to detect the activation of glial cells in the brain, inflammatory cell infiltration and proinflammatory cells. The expression level of factor TNF- alpha was changed in stress control group and normal animal group as control.
The results of the results of gene transcription showed that in the brain of the aged mice, TNF- alpha stimulated the significantly enhanced ICMA-1 gene transcription (P < 0.05), while IFN- gamma stimulation induced obviously enhanced MMP-12, SOCS-1 and SOCS-3 gene transcription (P < 0.05), and the transcriptional level of the TNFR2 gene was also significantly increased in the brain of the aged mice (P < 0.05). And the results of Western blotting showed that the expression of ICMA-1 protein induced by TNF- alpha stimulation was significantly enhanced in the old group (P < 0.05). The fluorescence double labeling results showed that the activated astrocytes and microglia were both ICAM-1. and green, compared with the middle aged group, TNF- alpha stimulated infiltration CD~ (3+), CD~ (4+) and CD~. Compared with stress control and normal group, the activation of astrocytes and microglia in the brain of REM sleep deprivation rats was obviously enhanced, and the expression of MHC- class II antigen and ED-2 positive macrophage increased obviously, and the expression level of TNF- alpha was obviously up (P < 0.0001) compared with the stress control and the normal group. Double labeled results showed that activated astrocytes, rather than microglia, express TNF- alpha up-regulated.
Conclusion 1. proinflammatory cytokines can amplify and prolong the function of pro-inflammatory response in the aging brain; 2. the normal senescent brain increases the immune response to inflammatory cytokines and increases the susceptibility. 3. the changes in sleep behavior can affect the activation and inflammatory response of glial cells in the brain and promote inflammatory cell causes. The child plays an important role in this process; 4. there is a very close relationship between the three proinflammatory cytokines, the aging brain inflammatory response and the changes in the sleep rhythm of the senescent individuals.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【相似文献】