臺灣博碩士論文加值系統

衛生福利部國人死因統計顯示，腦血管疾病為國人十大死因的第三位，造成社會及許多家庭的負擔。缺血性腦中風後，腦部功能急遽受損，但是神經功能再塑能力受限，導致患者後續神經功能性的回復緩慢，因此發展腦中風的治療以及神經保護劑實為當務之急。近來於離體及活體實驗中証實胸腺前素(prothymosin α)具有抗發炎及抗凋亡之神經保護能力，可以降低缺血性腦中風的損傷，然而胸腺前素是否具有神經重塑的能力仍需進一步探討。神經重塑對於神經功能的恢復相當重要，研究顯示於腦中風或腦組織損傷後的急性(約1-3天)和亞急性期(約7天)會表現不同蛋白參與神經細胞的分化、成長和受損修復。近期研究指出BDNF (brain-derived neurotrophy factor)可調控GAP-43 (growth-associated protein 43)與PSD-95 (post-synaptic density-95)表現，對於神經生長及神經重塑有促進作用。此外，神經分化會隨著突觸前蛋白synaptosomal-associated protein (SNAP-25)的表現量上升而增加。樹突小棘的密度在神經元受損後會大量減少造成神經元之間訊號傳導不良，因此提高樹突小棘生長密度在神經重塑及突觸重新連結是必須的。這些蛋白表現對於日後幫助腦神經功能的恢復與神經重塑扮演重要的角色。本實驗利用離體初代神經元細胞模式，探討胸腺前素在腦中風後之神經保護效果與神經再塑潛力以及其影響神經重塑中扮演關鍵性角色的蛋白之機制。根據本實驗結果顯示離體神經元細胞在受到oxygen-glucose deprivation (OGD)傷害下，胸腺前素對上述等神經重塑相關蛋白包括BDNF、SNAP-25、PSD-95和GAP-43表現有上調控的影響，並透過計算神經細胞軸突和樹突之生長和分支，認定胸腺前素確實會有效影響神經重塑的效果。胸腺前素為一非常有潛力之神經保護劑，在先前研究已証實對於缺血性腦中風有多種保護效果，本實驗接果顯示胸線前素具有神經重塑的效果，對於未來臨床上神經保護劑之研發提供新的方向。

Stroke, a disease with severe neurological impairment, is the main cause of death and often results in irreversible disability. Inflammatory responses initiated by stagnant blood flow after cerebral ischemia trigger the activation of immune cells and the release of inflammatory cytokines that contribute to the ischemic damage. Prothymosin α (ProT), a highly acidic nuclear protein, possesses multiple cellular functions. ProT has been shown to be a neuroprotective molecule due to its anti-necrotic and anti-apoptotic activities. However, it remains largely unclear whether ProT promotes neuroplasticity following ischemic cerebral stroke. To investigate the neuroplastic effect of ProT, we evaluated the levels of neuronal differentiation-associated proteins and the conditions of neurite outgrowth. An ischemic stroke model in vitro was established by using a primary cortical neuronal culture after oxygen-glucose deprivation (OGD). Our results reveal that ProT upregulate several neuroplasticity-associated proteins including brain-derived neurotrophic factor (BDNF), synaptosomal-associated protein-25 (SNAP-25), growth-associated protein-43 (GAP-43), and postsynaptic density protein-95 (PSD-95). Moreover, neurite outgrowth was measured by microtubule-associated protein (MAP-2) immunofluorescence staining show increasing dendrite outgrow with ProT treatment upon OGD injury. Collectively, our data indicate that ProT exerts potential neuroplastic effects.

Content
中文摘要-------------------------------------------------------------------------------------------I
Abstract------------------------------------------------------------------------------------------II
Acknowledgement--------------------------------------------------------------------------III
Contents----------------------------------------------------------------------------------------IV
List of Figures-------------------------------------------------------------------------------VII
List of Tables--------------------------------------------------------------------------------VIII
Abbreviation List----------------------------------------------------------------------------IX
Introduction------------------------------------------------------------------------------------1
Ischemic stroke---------------------------------------------------------------------------1
Cell death after ischemia---------------------------------------------------------------1
Neuroplasticity and associated protein---------------------------------------------1
Prothymosin α (ProT)-------------------------------------------------------------------2
Specific Aim------------------------------------------------------------------------------------4
Materials and methods---------------------------------------------------------------------5
Expression vectors----------------------------------------------------------------------5
Bacterial strain----------------------------------------------------------------------------5
Primers-------------------------------------------------------------------------------------5
Animal---------------------------------------------------------------------------------------5
Antibodies----------------------------------------------------------------------------------6
Reagents-----------------------------------------------------------------------------------7
Cell culture media------------------------------------------------------------------------7
Primary cortical neuronal culture-----------------------------------------------------8
Oxygen and glucose deprivation (OGD)-------------------------------------------8
Transfection of primary neuron cell--------------------------------------------------9
Immunofluorescence staining---------------------------------------------------------9
Measurement of neuronal dendrite branches and neuronal dendrite neurite outgrowth-------------------------------------------------------------------------9
RT-PCR analysis-----------------------------------------------------------------------10
Western Blotting------------------------------------------------------------------------10
Animal preparation, anesthesia and monitoring--------------------------------11
Experimental model and grouping-------------------------------------------------11
Neurobehavioral testing and body weight measurements-------------------12
Statistical analysis----------------------------------------------------------------------12
Results-----------------------------------------------------------------------------------------13
OGD injury decreased dendrite branches and length-------------------------13
OGD injury upregulated ProT expression----------------------------------------13
Transfection increased ProT gene expression----------------------------------13
Transfection of ProT gene increased dendritic arborization after OGD injury---------------------------------------------------------------------------------------14
Transfection of ProT gene increased neuroplasticity associated protein--------------------------------------------------------------------------------------------------14
ProT protein treatment increased dendritic arborization after OGD injury------------------------------------------------------------------------------------------------15
ProT protein treatment increased BDNF mRNA level after OGD injury--15
ProT protein treatment increased PSD95, GAP43, and SNAP25 protein level after OGD injury-----------------------------------------------------------------15
In vivo stroke animal model----------------------------------------------------------16
ProT improved neurologic behavior score---------------------------------------16
ProT increased plasticity-associated proteins at the penumbra------------17
ProT might improve neuroplasticity through GAP-43-------------------------17
Discussion------------------------------------------------------------------------------------18
Conclusion------------------------------------------------------------------------------------21
Figures-----------------------------------------------------------------------------------------22
Tables-------------------------------------------------------------------------------------------33
References------------------------------------------------------------------------------------35

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