【摘要】：背景及目的高遷移率族蛋白B1(High-mobility group box 1,HMGB1)是廣泛表達(dá)于真核細(xì)胞內(nèi)高度保守的一種非組氨酸核蛋白,參與基因的表達(dá)和核小體的定位。HMGB1參與了CNS疾病的發(fā)生發(fā)展,在腦卒中和脊髓損傷早期可作為炎癥因子加重神經(jīng)炎癥反應(yīng),后期則可促進(jìn)神經(jīng)血管的再生有利于神經(jīng)功能的恢復(fù)。在胚胎發(fā)育時(shí)期,HMGB1參與神經(jīng)元突觸的生長(zhǎng)及細(xì)胞的遷移。CNS損傷后,激活的星形膠質(zhì)細(xì)胞也可分泌HMGB1,但其對(duì)星形膠質(zhì)細(xì)胞遷移能力的影響還不清楚。前期的研究表明磁刺激能激活并調(diào)控星形膠質(zhì)細(xì)胞的遷移能力。本實(shí)驗(yàn)將進(jìn)一步研究MS誘導(dǎo)下HMGB1對(duì)星形膠質(zhì)細(xì)胞遷移的影響,并探討其作用機(jī)制。資料與方法1.星形膠質(zhì)細(xì)胞的提取、培養(yǎng)及鑒定:提取出生后為2-3天SD(Sprague-Dawley)大鼠大腦皮質(zhì)組織,分離培養(yǎng)星形膠質(zhì)細(xì)胞,并觀察其生長(zhǎng)及形態(tài),并用細(xì)胞免疫熒光技術(shù)觀察GFAP蛋白的表達(dá),鑒定星形膠質(zhì)細(xì)胞的純度。2.實(shí)驗(yàn)分組:根據(jù)不同頻率的磁刺激將實(shí)驗(yàn)分為3組:(1)對(duì)照組;(2)低頻磁刺激組(1HZ);(3)高頻磁刺激組(10HZ)。觀察細(xì)胞遷移能力,并運(yùn)用Western Blot觀察ERK1/2磷酸化及HMGB1表達(dá)情況。3.干擾HMGB1表達(dá)后細(xì)胞遷移實(shí)驗(yàn)分組:(1)控制組(磁刺激);(2)Si-RNA組(磁刺激+HMGB1 Si-RNA)。觀察細(xì)胞遷移能力,并運(yùn)用Western Blot檢測(cè)干擾效果。4.U0126阻滯后細(xì)胞遷移實(shí)驗(yàn)分組:(1)控制組(磁刺激);(2)實(shí)驗(yàn)組(磁刺激+U0126)。觀察細(xì)胞遷移能力,并運(yùn)用Western Blot觀察ERK1/2磷酸化及HMGB1表達(dá)情況。結(jié)果1.取培養(yǎng)的第3代大鼠星形膠質(zhì)細(xì)胞,用GFAP熒光染色鑒定,星形膠質(zhì)細(xì)胞純度95%。2.高頻磁刺激促進(jìn)星形膠質(zhì)細(xì)胞的遷移能力,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。且ERK1/2磷酸化及HMGB1的表達(dá)水平明顯高于對(duì)照組和低頻磁刺激組,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。3.HMGB1 Si-RNA干擾后,HMGB1表達(dá)水平顯著下降,且星形膠質(zhì)細(xì)胞的遷移能力相應(yīng)明顯下降,與控制組相比差異有統(tǒng)計(jì)學(xué)意義(P0.01)。4.U0126阻滯后,HMGB1表達(dá)水平顯著降低,且星形膠質(zhì)細(xì)胞的遷移能力相應(yīng)明顯下降,與控制組相比差異有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論1.高頻磁(10HZ)刺激促進(jìn)星形膠質(zhì)細(xì)胞的遷移,并可促進(jìn)ERK1/2的磷酸化。2.高頻磁(10HZ)刺激促進(jìn)HMGB1的表達(dá),并以自分泌的形式作用于星形膠質(zhì)細(xì)胞,參與磁刺激誘導(dǎo)的星形膠質(zhì)細(xì)胞遷移的過(guò)程。3.ERK1/2的磷酸化有利于HMGB1的表達(dá),HMGB1為ERK1/2的下游信號(hào)。
[Abstract]:Background and objective High mobility group protein B1 (High-mobility group box 1 HMGB1) is a highly conserved non-histidine nucleoprotein widely expressed in eukaryotic cells, which is involved in gene expression and nucleosome localization. HMGB1 is involved in the occurrence and development of CNS disease. In the early stage of stroke and spinal cord injury, it can be used as an inflammatory factor to aggravate the neuroinflammatory reaction, and to promote the regeneration of nerve vessels in the later stage is beneficial to the recovery of nerve function. During embryonic development, HMGB1 is involved in neuronal synaptic growth and cell migration. After CNS injury, activated astrocytes also secrete HMGB1, but its effect on astrocyte migration ability is unclear. Previous studies have shown that magnetic stimulation activates and regulates the migration of astrocytes. This study will further investigate the effects of HMGB1 on astrocyte migration induced by MS and its mechanism. Data and methods 1. Extraction, culture and identification of astrocytes: the cortical tissues of SD (Sprague-Dawley) rats were isolated and cultured for 2-3 days after birth, and the growth and morphology of astrocytes were observed. The expression of GFAP protein was observed by cell immunofluorescence technique, and the purity of astrocytes was identified. 2. 2. Experimental group: according to different frequency of magnetic stimulation, the experiment was divided into three groups: (1) control group, (2) low frequency magnetic stimulation group (1HZ); (3), high frequency magnetic stimulation group (10HZ). The ability of cell migration and the expression of ERK1/2 phosphorylation and HMGB1 were observed by Western Blot. After interfering with the expression of HMGB1, cell migration was divided into two groups: (1) control group (magnetic stimulation); (2) Si-RNA group (magnetic stimulation HMGB1 Si-RNA). The ability of cell migration was observed and the interference effect was detected by Western Blot. The cell migration after 4.U0126 arrest was divided into two groups: (1) control group (magnetic stimulation); (2) experimental group (magnetic stimulation U0126). Cell migration was observed and ERK1/2 phosphorylation and HMGB1 expression were observed by Western Blot. Result 1. The cultured astrocytes from the third passage of rats were identified by GFAP fluorescence staining. The purity of astrocytes was 95? 2. High frequency magnetic stimulation promoted the migration of astrocytes, and the difference was statistically significant (P0.01). The levels of ERK1/2 phosphorylation and HMGB1 expression were significantly higher than those of control group and low frequency magnetic stimulation group (P0.01). After 3.HMGB1 Si-RNA interference, the expression of HMGB1 decreased significantly. The migration ability of astrocytes was significantly decreased compared with the control group (P0.01). After 4.U0126 arrest, the expression of HMGB1 decreased significantly, and the migration ability of astrocytes decreased significantly. Compared with the control group, the difference was statistically significant (P0.01). Conclusion 1. High frequency magnetic (10HZ) stimulation promoted astrocyte migration and ERK1/2 phosphorylation. 2. 2. High frequency magnetic (10HZ) stimulation promoted the expression of HMGB1 and acted on astrocytes in the form of autocrine, which was involved in the process of the migration of astrocytes induced by magnetic stimulation. The phosphorylation of 3.ERK1/2 was beneficial to the expression of HMGB1. HMGB1 is the downstream signal of ERK1/2.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R651.2

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