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研究生: 陳郁淇
Chen, Yu-Chi
論文名稱: 惡性神經膠母細胞瘤所導致之腦水腫產生的影響
The Effects of Malignant Gliomas on the Development of Cerebral Edema
指導教授: 司君一
Sze, Chun-I
學位類別: 碩士
Master
系所名稱: 醫學院 - 細胞生物與解剖學研究所
Institute of Cell Biology and Anatomy
論文出版年: 2012
畢業學年度: 100
語文別: 中文
論文頁數: 56
中文關鍵詞: 腦水腫第四型水通道蛋白神經膠母細胞瘤血管性腦水腫細胞毒性腦水腫
外文關鍵詞: Brain edema, Aquaporin-4, Glioblastoma, Vasogenic edema, Cytotoxic edema
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  • 很多種類的腦部損傷都會產生腦水腫的情形,腦水腫的定義在於腦部組織水分增加,進而導致整個腦部體積增加,水通道蛋白-4 (Aquaporin-4,AQP4) 是中樞神經系統中最大量可控制水分進出的蛋白質。我的實驗主要在探討神經膠瘤所導致的腦水腫對於AQP4與血腦障壁(blood brain barrier,BBB) 的表現量及變化有什麼影響。
    實驗結果顯示在大腦乾溼重量分析中可以看出有打入腫瘤的大腦含水量有提升;在西方式點墨法和免疫組織染色法中可以發現在打入腫瘤細胞的右半腦的AQP4,膠質纖維酸性蛋白(glial fibrillary acidic protein, GFAP),缺氧誘導因子1α (Hypoxia-inducible factors1α,HIF1α) 表現量隨著時間的增加有漸漸上升的趨勢,且環繞在腫瘤周圍表現量越多,這個結果顯示血管性腦水腫(Vasogenic Edema)和細胞毒性腦水腫(Cytotoxic Edema)兩者皆有發生於腫瘤所導致的腦水腫模型;在AQP4和GFAP的雙重螢光染色實驗顯示AQP4會表現在原生的星狀神經膠細胞,小血管,微血管,活化和新生的星狀神經膠細胞,在不同的腦部損傷模型如Gliomatosis cerebrei,則新生的星狀神經膠細胞沒有發現相同的結果;GFAP和層黏蛋白 (laminin) 結果顯示血腦障壁上的基底膜和星狀神經膠細胞的末足分離;第一型血小板/內皮細胞附著因子 (Platelet/endothelial cell adhesion molecule-1,CD31) ,運鐵蛋白接受子 (transferrin receptor,CD71) ,血管內皮生長因子 (vascular endothelial growth factor,VEGF) 和血小板生長因子β(platelet-derived growth factor receptor-β,PDGFR-β) 的雙重螢光染色結果顯示腫瘤旁微血管的血腦障壁有崩解的情形產生。
    而根據這些實驗結果我們可以下一個結論:腫瘤所引起的腦水腫顯示細胞膜上的水通道蛋白與BBB結構的改變,甚至有腦部缺氧以及動物死亡的情形,本實驗動物模型將可以提供病理生理學重要的資訊用於探討惡性神經膠母細胞腫瘤旁水腫生成的原因。

    Cerebral edema causes increased brain volume with an absolute increase in cerebral tissue water content. Aquaporin-4 (AQP4) is one of the most common water channel membrane protein of central nervous system. To characterize the expression of AQP4 and blood brain barrier (BBB) changes in cerebral edema, we investigate rats’ brain with a glioma-induced cerebral edema.
    The water content was elevated in brain with a glioma-induced mass lesion than controls when measured by wet-dry method. Expression of AQP4, HIF1α, and glial fibrillary acidic protein(GFAP) proteins were increased in the right hemisphere surrounding the tumor which were demonstrated by Western blot and immunohistochemistry (IHC) staining. These observations suggest that tumor induced brain edema could have both vasogenic and cytotoxic edema components. The double immunofluorescence (IF) staining of AQP4 and GFAP showed that AQP4 were expressed in astrocytes, small blood vessels and capillaries, also in reactive astrocytes. These observations were not showed by IHC staining of human samples with reactive astrocytes due to different brain lesions such as Gliomatosis cerebrei. The expression of GFAP and laminin are not merged in tumor tissue, which indicating the astrocyte foot processes were released from basement membrane of BBB. The double immunofluorescence staining of CD31, CD71, VEGF, PDGFRβ showed that capillaries were disrupted in peritumoral area. These observations confirm that the brain tumor also affect BBB.
    In conclusion,the brain tumor-induced cerebral edema alters cell membrane water channel and blood brain barrier. It also causes cerebral ischemia and death. This animal model might provide useful information to study detail pathophysiological mechanisms in brain lesions that would cause edema due to different etiology.

    摘要........................................................Ⅰ Abstract ...................................................Ⅲ 致謝........................................................Ⅴ 目錄........................................................Ⅵ 緒論(Introduction).........................................1 腦水腫(Brain Edema)......................................2 腫瘤引發腦水腫(Brain tumor induces brain edema)..........2 多型性神經膠母細胞瘤(Glioblastoma multiforme,GBM).......3 三種腦水腫型態 (Three types of brain edema)..............4 細胞毒性水腫 (Cytotoxic Edema).......................4 血管性腦水腫(Vasogenic Edema)........................5 水通道蛋白(Aquaporin,AQPs)..............................6 中樞神經系統中的水通道蛋白(AQPs in CNS)..................7 血腦障壁 (Blood-brain barrier,BBB)........................8 研究動機與目的(Objective)................................9 方法與材料(Material & Method)................................11 細胞培養(Cell Culture).....................................12 腦水腫模型(Brain Edema Model)..............................12 動物準備(Animal Preparation)...........................12 大鼠腦定位(Rat Stereotactic Surgery)...................12 動物犧牲灌流 (Animal Perfusion)............................14 乾溼重分析(Wet-Dry Method).................................14 蛋白質純化(Protein Purification)...........................15 西方氏點默法(Western Blot).................................16 血腦障壁滲透度分析(BBB Permeability).......................16 組織分析(Histology Analysis)...............................17 H&E染色(H&E Staining)..................................18 免疫組織染色(Immunohistochemical staining,IHC).........19 雙重螢光染色(Double immunofluorescence staining,IF)....20 統計分析(Statistical analysis).............................20 實驗流程(Experiment Design)..................................23 實驗結果(Results)...........................................25 腦水腫型態(Brain Edema Morphology).........................26 腦水份測量(Brain Water Content Measurement).................26 腦水腫的標誌(Markers of Brain Edema).....................26 新生星狀膠細胞的表現(Expression of reactive astrocyte).....27 細胞毒性腦水腫的偵測(Detection of Cytotoxic Edema)........28 血腦障壁於腦水腫區域的通透性(BBB permeability in edema area).....28 腦水腫微血管的表現(Expression of capillary in hydrous brain).29 腦水腫區域的微血管型態(Capillary Morphology in Edema Area)..29 實驗討論(Discussion)........................................31 腦水腫使腦組織鬆散(Peritumoral edema loss brain neuropil)...32 腫瘤使腦部水分增加(Tumor increased brain water content).....32 腫瘤引起腦水腫隸屬的水腫類型(Peritumoral edema type).......32 AQP4於腫瘤引起腦水腫的意義(AQP4 in two types of brain edema).33 腫瘤引起腦水腫之原因(brain tumor causes brain edema)........34 腦水腫液之吸收(Brain edema fluid absorption)................35 AQP4於治療腦水腫之未來前景(AQP4&Treatment of brain edema)...36 實驗結論(Conclusion)........................................37 圖(Figure)..................................................39 參考文獻(Reference).........................................52

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