臺灣博碩士論文加值系統

哺乳動物中脊髓非常容易受到外力因素而損傷，脊髓損傷會造成
局部的缺血、離子的不平衡、麩氨酸的釋放以及去髓鞘化等種種退化
性傷害。先前報導指出脊髓損傷後給予生長因子，能夠有效達到血管
保護及促進寡突神經膠細胞新生的效果。雖然，脊髓損傷後的功能自
發性回復已經被發現，但其中所牽涉的因子及機制仍未被完整的研究
揭露出來。本實驗係採用250-300 公克的雄性大白鼠做動物模式，經
由300 毫克/公斤腹腔注射水化氯醛麻醉，將胸髓T8 左側橫切以模擬
脊髓損傷之用。實驗組動物分為 (1) 形態分析組以及 (2) 蛋白體分
析組。動物分別於損傷後第6 和24 小時，4、7、14 和21 天犧牲。動
物中樞神經組織分別以6% 三聚甲醛-離胺酸-過碘酸鹽固定，做為形
態學觀察，或另外以均質化做為蛋白質體分析之用。在光學顯微鏡下
觀察，可見損傷後白質及灰質損壞區域的變化，在高倍率光學顯微鏡
下觀察，可見損傷後細胞呈現空泡化的現象，在手術後第7 天，則可
顯著看見許多的梭狀纖維樣細胞散生於白質受損區之中。在電子顯微
鏡下觀察，受損區的神經細胞呈現細胞質消失、細胞膜以及有形結構
明顯被破壞，許多細胞呈現細胞凋亡的現象。細胞內空泡的超微結構，
呈現空泡狀或具有雙層單位膜的結構。脊髓損傷的後期，出現的類似
梭狀纖維樣細胞，而在其細胞質中可發現許多的粗糙內質網具有膨大
池狀結構。在蛋白體分析中，由西方墨漬法呈現血管內皮生長因子
(VEGF) 及細胞凋亡誘導因子 (AIF)，在損傷後期才有顯著表現；而
Bcl-2 則是在損傷後的急性期，即有大量表現並隨著回復時間的增加而
遞減。在損傷後蛋白體表現分析的二維凝膠電泳中，經由分析後定序
出數種不同的蛋白質體。本研究指出在損傷後的急性期中，Bcl-2 可能
扮演調控與保護的角色，而VEGF 則可能在脊髓損傷的後期具有保護
血管的功能。並且形態學的結果顯示，脊髓損傷後細胞死亡的方式不
僅含有細胞凋亡， 並且亦含有旁凋亡 (paraptosis) 與自噬凋亡
(autophagotic death) 的程式性死亡。

The adult mammalian central nervous system is vulnerable to various injuries or
trauma. Spinal cord injury (SCI) results in ischemia and hypoxia followed by a series of
degenerating events. SCI also results in ion unbalance, glutamate release and
demyelination. Administration of the growth factor leads to repair of blood vessels or
replacement of neurons and oligodendrocytes in the injured spinal cord. Spontaneous
functional recovery after spinal cord injury was found previously. However, the
mechanism involved in the spinal cord injury was still not fully elucidated. In this study,
adult male Sprague-Dawley rats (250-300g) were anesthetized with chloral hydrate (300
mg/Kg, i.p.) and hemisectioned (left side) at T8 vertebral level. In the experimental
group, animals were divided into two subgroups: (1) morphological analysis and (2)
proteomic analysis. Animals were sacrificed at 6, 12 hours, 1, 4, 7, 14 and 21 days after
spinal cord injury. Brain tissue either fixed with 6% pareformaldehyde-lysine-periodate
prepared for morphological analysis or homogenized for proteomic analysis. In the light
microscopic level, both white and gray matter were smashed and completely lesioned in
the experimental spinal cord. At higher magnification, abundant vacuoles were located
in the cytoplasm after spinal cord injury. At postlesion day 7, numerous spindle-shaped
fibrocyte-like cells occupied the injured area in the white matter of the spinal cord.
Under the electron microscope, the cell lost its cytoplasm and became amorphous
structure indicative of markedly destroyed cytoplasmic organelles. Many apoptotic-like
structures were also found. There were some vacuoles contain double membrane.
Several spindle-shaped fibrocyte-like cells contained abundant rough endoplasmic
reticulum and partially dilated cisternae. In the western blotting, VEGF and AIF were
markedly expressed at late stage of recovery but the Bcl-2 was expressed at early stage.
Numerous proteins were sequenced by LC/MS/MS followed 2-dimentional
electrophoresis. Our results indicate that Bcl-2 may protect the survival from cell death
at early stage and VEGF may protect the intact of the blood vessel during middle to late
stage after spinal cord injury. The morphological findings showed that programmed cell
death contained not only apoptosis but also paraptosis and autophagic death after injury.

中文摘要...........................................1
英文摘要...........................................2
前言...............................................3
材料與方法.........................................6果................................................17
討論..............................................23
參考文獻..........................................27
圖表與說明........................................36

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