磁刺激诱导下HMGB1对大鼠星形胶质细胞迁移影响的体外研究
发布时间：2018-11-13 18:08
【摘要】：背景及目的高迁移率族蛋白B1(High-mobility group box 1,HMGB1)是广泛表达于真核细胞内高度保守的一种非组氨酸核蛋白,参与基因的表达和核小体的定位。HMGB1参与了CNS疾病的发生发展,在脑卒中和脊髓损伤早期可作为炎症因子加重神经炎症反应,后期则可促进神经血管的再生有利于神经功能的恢复。在胚胎发育时期,HMGB1参与神经元突触的生长及细胞的迁移。CNS损伤后,激活的星形胶质细胞也可分泌HMGB1,但其对星形胶质细胞迁移能力的影响还不清楚。前期的研究表明磁刺激能激活并调控星形胶质细胞的迁移能力。本实验将进一步研究MS诱导下HMGB1对星形胶质细胞迁移的影响,并探讨其作用机制。资料与方法1.星形胶质细胞的提取、培养及鉴定:提取出生后为2-3天SD(Sprague-Dawley)大鼠大脑皮质组织,分离培养星形胶质细胞,并观察其生长及形态,并用细胞免疫荧光技术观察GFAP蛋白的表达,鉴定星形胶质细胞的纯度。2.实验分组:根据不同频率的磁刺激将实验分为3组:(1)对照组;(2)低频磁刺激组(1HZ);(3)高频磁刺激组(10HZ)。观察细胞迁移能力,并运用Western Blot观察ERK1/2磷酸化及HMGB1表达情况。3.干扰HMGB1表达后细胞迁移实验分组:(1)控制组(磁刺激);(2)Si-RNA组(磁刺激+HMGB1 Si-RNA)。观察细胞迁移能力,并运用Western Blot检测干扰效果。4.U0126阻滞后细胞迁移实验分组:(1)控制组(磁刺激);(2)实验组(磁刺激+U0126)。观察细胞迁移能力,并运用Western Blot观察ERK1/2磷酸化及HMGB1表达情况。结果1.取培养的第3代大鼠星形胶质细胞,用GFAP荧光染色鉴定,星形胶质细胞纯度95%。2.高频磁刺激促进星形胶质细胞的迁移能力,差异具有统计学意义(P0.01)。且ERK1/2磷酸化及HMGB1的表达水平明显高于对照组和低频磁刺激组,差异具有统计学意义(P0.01)。3.HMGB1 Si-RNA干扰后,HMGB1表达水平显著下降,且星形胶质细胞的迁移能力相应明显下降,与控制组相比差异有统计学意义(P0.01)。4.U0126阻滞后,HMGB1表达水平显著降低,且星形胶质细胞的迁移能力相应明显下降,与控制组相比差异有统计学意义(P0.01)。结论1.高频磁(10HZ)刺激促进星形胶质细胞的迁移,并可促进ERK1/2的磷酸化。2.高频磁(10HZ)刺激促进HMGB1的表达,并以自分泌的形式作用于星形胶质细胞,参与磁刺激诱导的星形胶质细胞迁移的过程。3.ERK1/2的磷酸化有利于HMGB1的表达,HMGB1为ERK1/2的下游信号。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2
本文编号：2329984
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2
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