自噬現象及其調控在脊髓損傷治療的應用研究

Autophagy and its manipulation for possible application in treatment of spinal cord injury

陳先志(Hsien-Chih Chen)

指導教授 ： 邱文達

臺北醫學大學/醫學院/臨床醫學研究所/博士(2012年)

https://doi.org/10.6831/TMU.2012.00220

摘要

脊髓損傷是一種對病患生理及心理具有嚴重破壞性影響的傷病，除了病患個人，對於其家庭和社會成本而言，實際上付出的代價也是非常巨大。雖然我們在了解脊髓損傷的病理生理機轉上已有顯著的進展，然而有關其引起後續細胞死亡的機制仍然不完全理解。細胞的死亡有三個主要類型，包括細胞壞死，細胞凋亡和細胞自噬。細胞壞死和細胞凋亡已知會在脊髓損傷以後發生，但細胞自噬這種細胞死亡方式則尚不清楚是否會發生。原發的衝擊損傷導致的細胞壞死是無法預防的，但在損傷震央附近的細胞雖倖免於最初的重創，卻可能會面臨繼發的生物化學影響所啟動的凋亡和自噬作用而導致細胞死亡。因此，本論文的主要目的，在於探討自噬現象在脊髓損傷後的發生與否，並期望藉由調控自噬現象來發展新的脊髓損傷治療對策。

在我們前段的研究，我們藉由西方墨點免疫印跡，免疫螢光和透射電子顯微鏡來證明自噬現象存在於大鼠脊髓損傷模型中。我們還發現了細胞自噬表現在實驗性大鼠脊髓損傷的時間過程，以及在急性期特定細胞的表現及其與細胞凋亡的關聯。鑒於一些研究已經提到了細胞自噬在神經退化性疾病和腦缺氧缺血引起的腦損傷中可能的保護作用，我們在後續的研究，假設在脊髓損傷後神經細胞的死亡，是可以經由調控細胞自噬的表現而加以避免。我們藉由西方墨點免疫印跡及大鼠運動功能評估，發現自噬現象是可以透過雷帕黴素抑制mTOR而使其表現增強，而且可以顯著地改善脊髓損傷大鼠運動功能的恢復；因此我們更深入探討雷帕黴素對大鼠脊髓損傷的影響，發現除了增進自噬表現以外，它在脊髓損傷模型中也有抗發炎反應及減少神經細胞損害的功能，因此而達到增進大鼠運動功能恢復的成果。

本研究藉由大鼠脊髓損傷模型印證自噬現象的存在，及其受調控後在脊髓損傷治療應用上的可能性，未來除了更深入探討細胞自噬與細胞凋亡的關聯，也可進一步發展藥物在外科手術治療後局部或靜脈給予，並參與有潛力的雞尾酒療法，以加速脊髓損傷臨床治療的進步。

關鍵字

脊髓損傷 ； 自噬 ； 雷帕黴素 ； 穿透式電子顯微鏡 ； 微管相關蛋白輕鏈3

並列摘要

Spinal cord injury (SCI) is a serious and debilitating health problem. Quite apart from the emotional and physical stress of SCI to the patient, the actual monetary costs of SCI to the family and community are enormous. Although remarkable progress has been made in pathophysiological mechanisms of SCI, the mechanisms causing cell deaths after SCI are still incompletely understood. There are three main types of cell death, including necrosis, autophagy and apoptosis. Cell deaths due to necrosis and apoptosis are known to occur after SCI; however, autophagic cell death is unclear. Prevention of cell necrosis before the primary injury is not possible. Cells around the epicenter of injuries that are spared from the initial trauma may experience a secondary biochemical insult to initiate the apoptosis and autophagic cell deaths. Therefore, the main purpose of this paper, is to explore the phenomenon of autophagy in spinal cord after injury or not, and hope that through manipulation of autophagy we can develop novel treatment for SCI.

In our first research, we depended on Western blotting, immunofluorescence, and transmission electron microscopy to prove autophagy expression exists in rat spinal cord injury model. We also found the time course of autophagy expression in experimental rat SCI, as well as autophagy expression in specific cells in the early stages and its correlation with apoptosis. As some researches in neurodegenerative diseases and brain hypoxia-ischemia, autophagy may induce neuroprotective effects on brain damage. We assumed death of neurons after SCI is possible avoided via manipulation of autophagy expression in the following study. We found rapamycin could increase autophagy expression by inhibition of mTOR through Western blots, and it was able to improve locomotor recovery in rats after SCI by way of BBB locomotor evaluation. We then in-depth explored the effects of rapamycin on SCI in rats. We found apart from promotion of autophagy, rapamycin has effects of anti-inflammation and reduction of neuronal damage, which leads to improvement of functional recovery.

Through this study in rats with SCI model proves existence of autophagy, and possibility for therapeutic application by manipulation of autophagy in SCI. In the future, we can further develop local or intravenous administration of drugs after surgical treatment, and participate in the potential cocktail therapy, to speed up the advancement of clinical therapy for spinal cord injury.

並列關鍵字

spinal cord injury ； autophagy ； rapamycin ； transmission electron microscopy ； microtubule-associated protein 3

參考文獻

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