【摘要】：目的:研究ESE1在脂多糖(LPS)誘導的大鼠中樞神經系統(tǒng)炎癥模型中的表達,并探討ESE1參與小膠質細胞活化以及神經元凋亡的機制。方法:1.整體水平,建立大鼠側腦室注射脂多糖(lipopolysaccharide,LPS)的神經炎癥模型;取54只Sprague-Dawley(SD)雄性大鼠,隨機分為兩組:LPS注射組(48只)和假手術組(6只)。通過蛋白質印跡法(Western Blot,WB)和免疫組織化學染色法(immunohistochemistry,IHC)檢測LPS注射后ESE1的表達和細胞類型定位變化。2.通過免疫熒光雙標記法(double-immunofluorescent staining,IF)檢測ESE1在LPS注射后與不同細胞的共定位情況。3.通過WB檢測LPS注射后i NOS、cleaved caspase-3、Bax及Bcl-2的表達,并通過IF分別檢測i NOS/Iba-1、i NOS/ESE1、Neu N/cleaved-capase-3、ESE1/cleaved-caspase-3的共定位情況,以明確ESE1與小膠質細胞活化及神經元凋亡的關系。4.細胞水平,建立脂多糖誘導的小膠質細胞活化模型,通過WB檢測i NOS以及ESE1的表達變化,并通過ELISA檢測促炎細胞因子的釋放,以及熒光素酶報告基因檢測NF-κB的轉錄活性確定ESE1對小膠質細胞活化的影響。同時,也構建小膠質細胞活化培養(yǎng)基(condition medium,CM)誘導的神經元凋亡模型,通過WB檢測cleaved-caspase3以及ESE1的表達變化,以及LDH釋放實驗確定ESE1對神經元凋亡的影響。結果:1.LPS側腦室注射后,大腦皮質ESE1表達上調,在1d后達到高峰,隨后降低。2.ESE1分別與小膠質細胞以及神經元共定位,并且ESE1與其共定位數(shù)目增加。3.LPS注射后,cleaved-caspase3、以及i NOS表達上調,達到峰值后,隨后下降;并且i NOS/Iba-1、i NOS/ESE1、Neu N/cleaved-capase-3、ESE1/cleaved-caspase-3定位增加。4.在小膠質細胞中敲低ESE1的表達能明顯降低i NOS的表達,以及促炎細胞因子的釋放,以及NF-κB的轉錄活性明顯降低。5.在神經元細胞中,敲低ESE1的表達能明顯降低CM引起的神經元中cleaved-caspase3以及Bcl-2的水平,同時降低CM引起的LDH釋放。結論:ESE1在LPS誘導的中樞神經系統(tǒng)中表達上調,其表達變化主要存在于小膠質細胞以及神經元中,而非星形膠質細胞。在中樞神經系統(tǒng)中,ESE1通過影響NF-κB的轉錄活性,影響小膠質細胞活化以及神經元的凋亡。
[Abstract]:Aim: to investigate the expression of ESE1 in rat model of central nervous system inflammation induced by lipopolysaccharide (LPS), and to explore the mechanism of ESE1 involved in the activation of microglia and neuronal apoptosis. Methods: 1. 54 male Sprague-Dawley (SD) rats were randomly divided into two groups: LPS injection group (n = 48) and sham operation group (n = 6). Western blotting (Western Blot,WB) and immunohistochemical staining (immunohistochemistry,IHC) were used to detect the expression of ESE1 and cell type localization after LPS injection. 2. Immunofluorescence double labeling (double-immunofluorescent staining,IF) was used to detect the co-localization of ESE1 with different cells after LPS injection. The expression of I NOS,cleaved caspase-3,Bax and Bcl-2 after LPS injection was detected by WB, and the co-localization of I NOS/Iba-1,i NOS/ESE1,Neu Nr / cleaved-capase-3 ESE1 / cleaved-caspase-3 was detected by IF. To clarify the relationship between ESE1 and microglia activation and neuronal apoptosis. 4. At cell level, lipopolysaccharide induced microglial activation model was established. The expression of I NOS and ESE1 was detected by WB, and the release of pro-inflammatory cytokines was detected by ELISA. The transcriptional activity of NF- 魏 B was determined by luciferase reporter gene. The effect of ESE1 on the activation of microglia was determined. At the same time, the neuronal apoptosis model induced by microglial activation medium (condition medium,CM) was also constructed. The expression of cleaved-caspase3 and ESE1 was detected by WB, and the effect of ESE1 on neuronal apoptosis was determined by LDH release assay. Results: after intracerebroventricular injection of 1.LPS, the expression of ESE1 in cerebral cortex increased, reached its peak after 1 day, then decreased. 2.ESE1 co-located with microglia and neurons, and the number of co-localization of ESE1 and ESE1 increased after 3.LPS injection. The expression of cleaved-caspase3, and I NOS were up-regulated, reached the peak and then decreased. And I NOS/Iba-1,i NOS/ESE1,Neu Nr / cleaved-capase-3 ESE1 / cleaved-caspase-3 locus increased by 4. 4%. The expression of knockout ESE1 in microglia decreased the expression of I NOS, the release of proinflammatory cytokines and the transcription activity of NF- 魏 B. In neuronal cells, the low expression of ESE1 could significantly decrease the levels of cleaved-caspase3 and Bcl-2 in neurons induced by CM, and decrease the release of LDH induced by CM. Conclusion: the expression of ESE1 is up-regulated in the central nervous system induced by LPS, and the expression changes are mainly in microglia and neurons, but not astrocytes. In the central nervous system, ESE1 affects the activation of microglia and the apoptosis of neurons by affecting the transcriptional activity of NF- 魏 B.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R741

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