系統識別號 U0026-2407201413102700
論文名稱(中文) 探討脊髓損傷後引發星狀膠質細胞中CEBPD活化所扮演之角色
論文名稱(英文) Investigation of the roles of CCAAT/enhancer binding protein delta (CEBPD) in astrogliosis after spinal cord injury
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 102
學期 2
出版年 103
研究生(中文) 邱乃恩
研究生(英文) Nai-En Chiu
學號 S26014112
學位類別 碩士
語文別 英文
論文頁數 70頁
口試委員 指導教授-王育民
中文關鍵字 脊髓損傷  星狀膠質細胞增生  神經膠質疤痕  MMP3  RhoA 
英文關鍵字 spinal cord injury  astrogliosis  glial scar  MMP3  RhoA 
中文摘要 脊髓損傷是一種常見且具破壞性的中樞神經系統疾病,其會導致脊髓中結構的瓦解並伴隨著有限的神經元再生乃至於後續的功能恢復降低的情形發生。脊髓損傷都會伴隨著星狀膠質細胞增生的產生,而星狀膠質數目大量的增加並活化為星狀膠質細胞增生的特徵。在嚴重的損傷之下,星狀膠質細胞增生會形成無法逆轉的神經膠質疤痕進而成為神經再生的屏障。因此,探討星狀膠質細胞如何被活化而形成神經膠質疤痕,將有助於脊髓損傷的治療。CEBPD是一個轉錄因子,其可受發炎因子TNF-α 和 IL-1β調控,而且在許多發炎相關疾病中,例如阿茲海默症病人中發現CEBPD在星狀膠質細胞有過度表現的現象。本研究發現在老鼠脊髓損傷的切片觀察到CEBPD會大量表現於神經膠質疤痕中。進一步地,我們對小鼠在脊髓損傷後星狀膠質細胞中存在CEBPD與否的影響進行行為分析,我們發現CEBPD的缺失有助於脊髓損傷小鼠的行動恢復。在分子機制探討中,星狀膠質細胞在IL-1β處理下,CEBPD的增加會經由抑制RhoA的路徑,而抑制了自身細胞的移動。有趣的是,在表現CEBPD的星狀膠質細胞會產生分泌型因子MMP3以促進星狀膠質細胞的移動。總結以上結果,我們推論出,在脊隨髓損傷區的星狀膠質細胞會因CEBPD的活化,而直接於損傷位置固著並釋放出促移行因子MMP3,而致使較遠端的未活化之星狀膠質細胞的移行至受損區,再被進一步激活並參與神經膠質疤痕的形成。
英文摘要 Spinal cord injury (SCI) is a common and devastating central nervous system (CNS) disease that results in disruption of cord microstructure and is followed by limited neuronal regeneration and functional recovery impairment. After SCI, astrocytes, the most abundant glial cells in the CNS, become reactive and hypertrophy. Astrogliosis is an increase in the number of astrocytes to above-normal levels that can be observed in all CNS injuries and neuroinflammatory diseases. In severe cases of injury, astrogliosis results in the formation of irreversible glia scarring that acts as a regeneration barrier. Thus, investigation targeting on astrocyte activation and glial scar formation could be useful for SCI therapy. Transcription factor CCAAT/enhancer binding protein delta (CEBPD) is responsive to inflammatory factors such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) and has been observed in many inflammation-related diseases including AD. In SCI mice, our results showed that CEBPD is expressed in reactive astrocyte border. Using animal behavior tests, we found that a better recovered effect was observed in injured Cebpd-deficient mice. Our results showed that increase of CEBPD in astrocytes inhibited their self-migration ability through the RhoA pathway upon IL-1β treatment. In addition, the conditioned medium of astrocyte expressing CEBPD could promote the migration of inactive astrocytes. We further identified matrix metalloproteinase-3 (MMP-3) was responsive to CEBPD in astrocytes through a transcriptional regulation. Taken together, the results suggested that the migration inactive astrocytes can be promoted by MMP3 secreted from the fixed activated astrocyte expressing CEBPD, which contributes to the formation of glial scar in SCI.
論文目次 Contents
Abstract I
Abstract in Chinese III
Acknowledgments V
Contents VII
Chapter 1 Introduction 1
1.1 Spinal cord injury 1
1.2 Inflammation following spinal cord injury 2
1.3 Astrogliosis and glial scar formation 3
1.4 CCAAT/enhancer-binding protein delta (CEBPD) 3
1.5 Matrix metalloproteinases (MMPs) family 4
1.6 Ras homolog gene family, member A (RhoA) 5
1.7 Motivation 5
Chapter 2 Materials and methods 7
2.1 Materials 7
2.2 Methods 7
Cell culture and isolation of primary mouse astrocytes 7
Quantitative PCR (Q-PCR) 8
Western blot analysis 8
Luciferase reporter assay 9
Lentiviral knockdown 9
Immunofluorescence analysis 10
Cell migration assays 11
Spinal cord injury mouse model 11
Assessment of recovery 11
Open-field locomotion. 12
Rotarod 12
Footprint analysis. 12
Luxol fast blue assay 12
Chapter 3 Results 14
3.1 CEBPD expression in astrocytes associates with glial scar formation after SCI 14
3.2 Loss of Cebpd improves behavioral recovery in SCI mice 14
3.3 Loss of CEBPD shows a decreased glial scar formation and increased neuronal regeneration 15
3.4 CEBPD has no effect on the proliferation of astrocytes in glial scar formation after SCI 15
3.5 Increasing of CEBPD expression in astrocytes attenuates self-migration through RhoA inhibition 16
3.6 MMP3 contributes to promote the migration of inactive astrocytes in conditioned medium of astrocyte expressing CEBPD 17
3.7 Conclusions 18
Chapter 4 Discussion 20
4.1 CNS injury and CEBPD 20
4.2 Glial scar formation and CEBPD 21
4.3 Glial scar formation and MMP3 23
4.4 CEBPD for SCI novel therapeutic target 24
References 26
Figures 35
Figure 1. CEBPD expression in astrocytes associates with glial scar formation in SCI mice. 36
Figure 2. Loss of CEBPD shows a recovery effect in SCI mice 38
Figure 3. Loss of CEBPD attenuates glial scar formation and is more permeable for axon passing through in SCI mice. 40
Figure 4. Cebpd has no effect on the proliferation of astrocytes. 42
Figure 5. Cebpd contributes to IL-1β-inhibited astrocytes migration. 43
Figure 6. Cebpd represses RhoA transcription in IL-1β-treated primary astrocytes. 44
Figure 7. Increase of CEBPD down-regulates RhoA expression in astrocytes in SCI mice. 45
Figure 8. Conditioned media from astrocytes expressing CEBPD induces the migration of inactive astrocytes. 46
Figure 9. CEBPD positively regulates Mmp3 transcription in primary astrocytes 48
Figure 10. Mmp3 expression was attenuated in the astrocytes of spinal cord injured-Cebpd-/- mice. 49
Figure 11. Conditioned media from MMP3-knockdowned astrocytes inhibits the migration of inactive astrocytes. 50
Appendixes 51
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