在成熟的中樞神經系統中，有限的神經再生仍舊是現今醫學界的一大挑戰。中樞神經系統受傷之後，再生相關基因不容易被活化，且環境中會出現抑制性分子，降低神經細胞存活率並抑制神經再生。因為中和環境抑制分子對於神經再生的幫助有限，實際上可幫助中樞神經再生的作法為誘導再生相關基因再次活化。組蛋白脫乙醯酶(簡稱HDACs)及組蛋白乙醯轉移酶(簡稱HATs)在組蛋白的修飾上扮演重要的角色，進而調節再生相關基因的表現。P300是一種參與神經系統發育的HAT，但未知其是否能促進大鼠皮質神經細胞的再生。在本篇論文中，我利用從18天大的胎鼠所取出的皮質神經細胞來做實驗，分別加入HDAC的抑制劑Trichostatin A (TSA)及HAT的抑制劑 Anacardic acid (AA)，觀察神經細胞受傷後的再生。初步的結果顯示，TSA能增加組蛋白H3的乙醯化，但沒有促進神經再生的效果。AA則降低組蛋白H3和alpha-tubulin的乙醯化，減少WNT3A的mRNA表現，但不能促進神經再生。另外，在皮質神經細胞中高度表現P300會增加WNT3A的mRNA表現，但無助於神經再生。雖然TSA、AA和高度表現P300會影響到神經細胞內組蛋白的乙醯化或WNT3A的mRNA表現，但並不會促進大鼠皮質神經細胞的再生。

Limited neuronal regeneration is still a challenge for adult central nervous system (CNS). Regeneration-associated genes (RAGs) are difficult to be activated in CNS. There are many inhibitory molecules existing after injury. These molecules decrease neuronal survival and inhibit neuronal regeneration. Since neutralization of the extracellular molecules allows only limited axonal regeneration, one practical approach to promote regeneration of injured adult CNS neurons is to induce re-expression of RAGs. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are enzymes playing critical roles in histone modification. P300 is a HAT that involves in neuronal differentiation, but whether P300 promotes regeneration of injured rat cortical neurons is unknown. In this thesis, we used E18 rat cortical neurons as a cell model. HDAC inhibitor Trichostatin A (TSA) and HAT inhibitor Anacardic acid (AA) were added respectively to cortical neurons before scraping injury. Results showed that TSA increased the acetylation of histone H3 but has no effect on promoting neurite re-growth of rat cortical neurons. On the other hand, AA decreased the acetylation of histone H3, the acetylation of alpha-tubulin and the mRNA level of WNT3A. Yet, AA did not promote neurite re-growth. In addition, overexpression of P300 increased the mRNA level of WNT3A but did not promote neurite re-growth. Although TSA, AA and overexpression of P300 affected the acetylation of histone or the mRNA level of WNT3A, these treatments did not promote neuronal regeneration of rat cortical neurons.

Abstract...I
摘要...II
誌謝...III
Index...V
Abbreviations...VIII
Introduction...1
The nervous systems...1
Regeneration-associated genes...1
Limits of neuronal regeneration in CNS...2
Gene expression regulated by HATs and HDACs...3
The effects of HATs and HDACs in the nervous system...4
The effects of HAT and HDAC inhibitors...5
Function of P300 on histone modification...6
Materials and Methods...8
Antibodies and reagents...8
Primary cortical neuron cell culture...9
Preparation of cell lysate...9
Western blotting...10
Immunofluorescence staining...11
Transfection...11
Analysis of neurite re-growth...12
Injury assay and gap closure...12
RNA extraction...12
Reverse transcription (RT)...13
Real-time polymerase chain reaction (Q-PCR)...13
Statistical analysis...14
Results...15
TSA induces the acetylation of histone H3 in injured cortical neurons...15
TSA has no effect of promoting neurite re-growth of injured cortical neurons...16
AA decreases the acetylation of histone H3 in injured cortical neurons...16
AA does not enhance neurite re-growth of injured cortical neurons...17
AA (5 μM) does not enhance neurite re-growth of injured cortical neurons...18
AA compromises the acetylation of alpha-tubulin during regeneration of injured cortical neurons...18
AA inhibits the expression of WNT3A during regeneration of injured cortical neurons...19
The mRNA level of P300 does not change during regeneration of injured cortical neurons...19
The protein level of P300 decreases during regeneration of injured cortical neurons...20
Overexpression of P300 does not enhance neurite re-growth of injured cortical neurons...21
Overexpression of p300 increases the mRNA level of WNT3A of injured cortical neurons on DIV8...21
Discussion...23
Figures...25
Figure 1. Pre-treatment of TSA induces the acetylation of histone H3 in injured cortical neurons....25
Figure 2. Pre-treatment of TSA has no effect on neurite re-growth of injured cortical neurons...28
Figure 3. Pre-treatment of AA decreases the acetylation of histone H3 in injured cortical neurons...29
Figure 4. Pre-treatment of AA dose not enhance neurite re-growth of injured cortical neurons...32
Figure 5. Pre-treatment of 5 μM AA does not enhance neurite re-growth of injured cortical neurons in 24 hours...34
Figure 6. AA decreases of the acetylation of alpha-tubulin during regeneration of injured cortical neurons...35
Figure 7. AA treatment decreases mRNA level of WNT3A during regeneration of injured cortical neurons...36
Figure 8. The mRNA level of P300 remains constant during regeneration of injured cortical neurons...37
Figure 9. The protein level of P300 decreases during regeneration of injured cortical neurons...38
Figure 10. Overexpressing P300 does not enhance neurite re-growth of injured cortical neurons...41
Figure 11. Overexpression of P300 enhances the mRNA level of WNT3A in injured cortical neurons on DIV8...43
Appendix...44
Figure A1. Determination of TSA concentration on neurite re-growth...45
Figure A2. Determination of AA concentration on neurite re-growth...47
Tables...48
Table 1. P300 Plasmids for transfection...48
Table 2. Sequences of the Q-PCR primers used in this thesis...49
References...50

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