| Ski 在大鼠星形胶质细胞炎症活化中的作用研究 | |
| Alternative Title | Study on the role of Ski in the inflammatory activation of rat astrocytes |
| 王明 | |
| Subtype | 硕士 |
| Thesis Advisor | 张海鸿 |
| 2019-03-08 | |
| Degree Grantor | 兰州大学 |
| Place of Conferral | 兰州 |
| Degree Name | 硕士 |
| Degree Discipline | 外科学 |
| Keyword | Ski 星形胶质细胞 炎症因子 脂多糖 RNA 干扰 |
| Abstract | 目的：Ski作为一种进化保守蛋白，广泛分布于不同的组织和细胞，涉及多种细胞过程。前期研究发现，Ski在脊髓损伤后反应性星形胶质细胞的增殖和迁移过程中发挥重要的调节作用。星形胶质细胞的炎症活化是其增殖和迁移的重要因素。然而，Ski在星形胶质细胞炎症活化中的作用尚不清楚。本实验研究目的是分析Ski在星形胶质细胞介导的炎症反应中的作用，及其对星形胶质细胞活化和炎症因子释放的影响。方法：取新生3d内Sprague-Dawley大鼠大脑皮质分离星形胶质细胞，体外培养。使用GFAP抗体进行免疫荧光染色鉴定纯度后，用1μg/ml脂多糖（lipopolysaccharide，LPS）诱导星形胶质细胞建立炎症模型，通过Western blot技术观察Ski、GFAP和iNOS蛋白表达变化，ELISA方法检测细胞培养上清中TNF-α、IL-1β 和IL-6 释放水平。然后将细胞分为空白对照组、阴性对照组、Ski-siRNA组，观察Ski沉默后对LPS诱导星形胶质细炎症活化的影响。结果：免疫荧光染色鉴定星形胶质细胞纯度大于98%，符合实验要求。给予LPS刺激星形胶质细胞后，Ski在LPS诱导的星形胶质细胞中升高，呈时间依耐性。此外，LPS作用于星形胶质细胞24h后，星形胶质细胞活化标志蛋白（GFAP）及炎症细胞因子（TNF-α、IL-1β、IL-6和iNOS)表达上调。为检测Ski在星形胶质细胞炎症活化中的具体作用，我们通过siRNA转染实验证实沉默Ski可以抑制炎症细胞因子（TNF-α、IL-1β、IL-6和iNOS)产生、GFAP表达和NF-κB核转位。结论：LPS可诱发大鼠星形胶质细胞活化及炎症反应。沉默Ski能够抑制LPS诱导星形胶质细胞活化和炎症细胞因子的分泌，表明Ski参与调节LPS诱导星形胶质细胞的炎症活化。 |
| Other Abstract | Objective: Ski, an evolutionarily conserved protein, is widely distributed in different tissues and cells and involved in various cellular processes. Previous studies have found that Ski plays an important regulatory role in the proliferation and migration of reactive astrocytes after spinal cord injury. Inflammatory activation of astrocytes is an important factor in their proliferation and migration. However, the role of Ski in activation and inflammatory responses of astrocytes is unclear. The purpose of experimental study was to analyze the role of Ski in astrocyte-mediated inflammatory responses and its effects on astrocyte activation and inflammatory cytokine release. Methods: Astrocytes were isolated from the cerebral cortex of 1-3-day-old Sprague-Dawley rats and cultured in vitro. The purity of astrocytes was confirmed by immunofluorescence staining of glial fibrillary acidic protein (GFAP) antibody, Inflammatory models were induced by 1 μg/ml lipopolysaccharide (LPS) in astrocytes. The expressions of Ski, GFAP and iNOS were observed by Western blot. The release levels of TNF-α, IL-1β and IL-6 were detected by ELISA. The cells were divided into control group, non-silencing group and Ski-siRNA group. Then the effects of Ski silencing on LPS-induced inflammatory activation in astrocytes were observed.Results: The purity of astrocytes was more than 98% by immunofluorescence staining, which met the experimental requirements. After astrocytes were stimulated by LPS, Ski was increased by LPS in a time-dependent manner. Moreover, the expression of astrocyte activation marker protein (GFAP) and Proinflammatory cytokines (TNF-α, IL-1β, IL-6 and iNOS) was up-regulated after LPS acted on astrocytes for 24 h. To study the concrete role of Ski in astrocyte inflammatory response, we confirmed that silencing of Ski can inhibit the production of inflammatory cytokines (TNF-α, IL-1β, IL-6 and iNOS), GFAP expression and NF-κB nuclear translocation by siRNA transfection experiments. Conclusion: LPS can induce rat astrocyte activation and inflammatory response. Silencing of Ski can inhibit the activation of astrocytes and the secretion of inflammatory cytokines by LPS, which demonstrate that Ski may be involved in the regulation of LPS-induced inflammatory activation of astrocytes. |
| Pages | 51 |
| URL | 查看原文 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | https://ir.lzu.edu.cn/handle/262010/338671 |
| Collection | 第二临床医学院 |
| Affiliation | 第二临床医学院 |
| First Author Affilication | Second Clinical School |
| Recommended Citation GB/T 7714 | 王明. Ski 在大鼠星形胶质细胞炎症活化中的作用研究[D]. 兰州. 兰州大学,2019. |
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