磁刺激通过PEA-15对星形胶质细胞迁移的影响及机制

发布时间：2018-01-03 08:40

本文关键词：磁刺激通过PEA-15对星形胶质细胞迁移的影响及机制　出处：《郑州大学》2016年硕士论文　论文类型：学位论文

更多相关文章： 磁刺激 星形胶质细胞 PEA-15 siRNA

【摘要】：背景及目的星形胶质细胞磷酸化蛋白PEA-15(Phosphoprotein enriched in astrocytes-15k Da)广泛存在于细胞质,位于常染色体Iq21-22,其含有一个DED结构域(death effector domain,DED)---死亡效应区,抑制细胞凋亡。ERK1/2(Extracellular signal-regulated kinases)参与细胞增殖、存活和细胞迁移等多种细胞进程。磁刺激可通过激活ERK通路,促进星形胶质细胞的迁移,并对中枢神经损伤后的炎症反应等起到重要的神经保护作用。PEA-15通过结合ERK1/2阻止其在细胞核中聚集,调控EKR1/2功能,其与磁刺激对星形胶质细胞的迁移作用密切相关。本研究主要是在体外实验中观察磁刺激是否可通过调节PEA-15影响ERK1/2通路,调控星形胶质细胞的迁移作用,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、细胞免疫荧光鉴定:解剖新生1-3天SD(Sprague-Dawley)大鼠获取大脑皮质组织,运用差速贴壁技术分离培养,获取星形胶质细胞,并观察其形态及生长,然后应用免疫荧光技术鉴定GFAP蛋白的表达。2.实验分组:体外划痕试验,根据不同的磁刺激强度将实验分为3组:(1)对照组(不给予磁刺激,但细胞同其他刺激组置于同一环境中);(2)A组(给予30%最大磁刺激强度);(3)B组(给予60%最大磁刺激强度)。3.干扰PEA-15表达后划痕试验实验分组:(1)A组(不给予磁刺激,进行阴形si RNA转染);(2)B组(不给予磁刺激,但对细胞进行PEA-15 si RNA转染,干扰PEA-15表达);(3)C组(给予磁刺激,进行阴性si RNA转染);(4)D组(给予磁刺激,同时进行si RNA转染,干扰PEA-15表达)。观察划痕愈合情况,并运用Western Blot观察PEA-15及其蛋白磷酸化的表达变化。利用荧光测定转染效率;Western Blot半定量测定干扰效果。结果1.从新生1-3天大鼠脑皮质中提取,经差速贴壁分离、培养的星形胶质细胞,细胞免疫荧光鉴定胶质纤维酸性蛋白GFAP表达阳性,DAPI染核测定细胞纯度较高。2.在体外划痕实验中发现,随着磁刺激强度的增加,划痕面积愈合越快,在30%-60%最大磁刺激强度的强度下,星形胶质细胞迁移速度和强度呈正相关,与对照组相比差异具有统计学意义(P0.05)。3.通过细胞转染荧光提示该转染试剂具有较高的转染效率,对转染细胞进行PEA-15蛋白的测定,Western Blot结果提示si RNA270具有最佳的干扰效果。划痕实验中转染组与磁刺激组均较正常组迁移明显。磁刺激组的P-PEA-15增加明显。结论1.差速贴壁结合摇床方法可培养纯度较高的高星形胶质细胞。2.体外划痕实验中,在磁刺激30%-60%的强度下可明显促进星形胶质细胞的迁移。3.干扰PEA-15的表达后可明显促进星形胶质细胞迁移,磁刺激后可能通过磷酸化PEA-15而促进星形胶质细胞迁移。
[Abstract]:Background and objective PEA-15(Phosphoprotein enriched in astrocytes-15k Da). It is widely found in the cytoplasm. It is located on autosomal Iq21-22 and contains a DED domain death effector domain-death effector region. Inhibition of apoptosis. ERK1 / 2 extracellular signal-regulated kineses were involved in cell proliferation. Magnetic stimulation can promote astrocyte migration by activating ERK pathway. It also plays an important neuroprotective role in the inflammatory response after central nervous system injury. PEA-15 inhibits its aggregation in the nucleus and regulates the function of EKR1/2 by binding with ERK1/2. It is closely related to the effect of magnetic stimulation on the migration of astrocytes. This study is mainly to investigate whether magnetic stimulation can affect the ERK1/2 pathway by regulating PEA-15 in vitro. Regulating the migration of astrocytes and exploring its mechanism. Data and methods 1. Extraction and culture of astrocytes. Cellular immunofluorescence identification: the cerebral cortex was obtained from the SD Sprague-Dawley rats at 1-3 days after dissection. Astrocytes were isolated and cultured by differential adherent technique. The morphology and growth of GFAP protein were observed, and then the expression of GFAP protein was identified by immunofluorescence technique. 2. Experiment group: in vitro scratch test. According to the different intensity of magnetic stimulation, the experiment was divided into 3 groups: control group (not given magnetic stimulation, but the cells were placed in the same environment as other stimulation groups); Group A (given 30% maximum magnetic stimulation intensity); Group B (60% maximum intensity of magnetic stimulation). Scratch test after interfering with PEA-15 expression was divided into two groups: group A (no magnetic stimulation, negative si RNA transfection); Group B (no magnetic stimulation), but the cells were transfected with PEA-15 si RNA, which interfered with the expression of PEA-15. Group C was given magnetic stimulation and transfected with negative si RNA. Group D was treated with magnetic stimulation and si RNA transfection, which interfered with the expression of PEA-15. The healing of scratches was observed. Western Blot was used to observe the expression of PEA-15 and its protein phosphorylation, and the transfection efficiency was measured by fluorescence. Results 1. The astrocytes were isolated from the cortex of neonatal 1-3 day rats and separated by differential adhesion. 2. Cellular immunofluorescence was used to detect the purity of glial fibrillary acidic protein (GFAP) GFAP positive cells. 2. In vitro scratch test, it was found that the intensity of magnetic stimulation increased with the increase of magnetic stimulation intensity. The faster the scratch area healed, the more positive correlation was between the speed of astrocyte migration and the intensity of astrocyte migration under the maximum magnetic stimulation intensity of 30% to 60%. Compared with the control group, the difference was statistically significant (P 0.05). The fluorescence of the transfected cells indicated that the transfection reagent had a higher transfection efficiency, and the PEA-15 protein of the transfected cells was determined. Western Blot results suggest si. In the scratch test, the migration of P-PEA-15 in the transfected group and the magnetic stimulation group was significantly higher than that in the normal group, and the P-PEA-15 level in the magnetic stimulation group was significantly increased. Conclusion 1. High purity astrocytes. 2. In vitro scratch test. Magnetic stimulation of 30- 60% can significantly promote the migration of astrocytes. 3. Interfere with the expression of PEA-15 can significantly promote the migration of astrocytes. Magnetic stimulation may promote astrocyte migration through phosphorylation of PEA-15.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R741

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