【摘要】：目的:研究ESE1在脂多糖(LPS)誘導(dǎo)的大鼠中樞神經(jīng)系統(tǒng)炎癥模型中的表達(dá),并探討ESE1參與小膠質(zhì)細(xì)胞活化以及神經(jīng)元凋亡的機(jī)制。方法:1.整體水平,建立大鼠側(cè)腦室注射脂多糖(lipopolysaccharide,LPS)的神經(jīng)炎癥模型;取54只Sprague-Dawley(SD)雄性大鼠,隨機(jī)分為兩組:LPS注射組(48只)和假手術(shù)組(6只)。通過蛋白質(zhì)印跡法(Western Blot,WB)和免疫組織化學(xué)染色法(immunohistochemistry,IHC)檢測(cè)LPS注射后ESE1的表達(dá)和細(xì)胞類型定位變化。2.通過免疫熒光雙標(biāo)記法(double-immunofluorescent staining,IF)檢測(cè)ESE1在LPS注射后與不同細(xì)胞的共定位情況。3.通過WB檢測(cè)LPS注射后i NOS、cleaved caspase-3、Bax及Bcl-2的表達(dá),并通過IF分別檢測(cè)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(zhì)細(xì)胞活化及神經(jīng)元凋亡的關(guān)系。4.細(xì)胞水平,建立脂多糖誘導(dǎo)的小膠質(zhì)細(xì)胞活化模型,通過WB檢測(cè)i NOS以及ESE1的表達(dá)變化,并通過ELISA檢測(cè)促炎細(xì)胞因子的釋放,以及熒光素酶報(bào)告基因檢測(cè)NF-κB的轉(zhuǎn)錄活性確定ESE1對(duì)小膠質(zhì)細(xì)胞活化的影響。同時(shí),也構(gòu)建小膠質(zhì)細(xì)胞活化培養(yǎng)基(condition medium,CM)誘導(dǎo)的神經(jīng)元凋亡模型,通過WB檢測(cè)cleaved-caspase3以及ESE1的表達(dá)變化,以及LDH釋放實(shí)驗(yàn)確定ESE1對(duì)神經(jīng)元凋亡的影響。結(jié)果:1.LPS側(cè)腦室注射后,大腦皮質(zhì)ESE1表達(dá)上調(diào),在1d后達(dá)到高峰,隨后降低。2.ESE1分別與小膠質(zhì)細(xì)胞以及神經(jīng)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(dá)上調(diào),達(dá)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(zhì)細(xì)胞中敲低ESE1的表達(dá)能明顯降低i NOS的表達(dá),以及促炎細(xì)胞因子的釋放,以及NF-κB的轉(zhuǎn)錄活性明顯降低。5.在神經(jīng)元細(xì)胞中,敲低ESE1的表達(dá)能明顯降低CM引起的神經(jīng)元中cleaved-caspase3以及Bcl-2的水平,同時(shí)降低CM引起的LDH釋放。結(jié)論:ESE1在LPS誘導(dǎo)的中樞神經(jīng)系統(tǒng)中表達(dá)上調(diào),其表達(dá)變化主要存在于小膠質(zhì)細(xì)胞以及神經(jīng)元中,而非星形膠質(zhì)細(xì)胞。在中樞神經(jīng)系統(tǒng)中,ESE1通過影響NF-κB的轉(zhuǎn)錄活性,影響小膠質(zhì)細(xì)胞活化以及神經(jīng)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(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R741

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