臺灣博碩士論文加值系統

微小膠細胞屬於大腦裡的一種防禦性細胞，其主要功能是保護大腦避免受到外來物質的侵襲，與維持大腦裡微環境之恆定。有許多研究指出周邊發炎會引發大腦微小膠細胞活化，如：細胞體腫脹、肥大，突觸的縮短、肥大，表現特殊蛋白質，分泌細胞激素等。當中樞神經系統持續處在發炎的情況下，與許多的神經退化性疾病息息相關，包括帕金森氏症、阿茲海默氏症及多重硬化症等。先前的文獻指出微小膠細胞的密度在腦中是呈現不均勻的分佈；但此一分佈不均勻的情形是否會影響周邊發炎後微小膠細胞活化的情形則仍不明瞭。因此本研究的目的是比較周邊發炎後腦中各區域微小膠細胞活化之情形。首先，以Iba-1來標示微小膠細胞並計算小鼠各腦區中微小膠細胞的密度發現微小膠細胞的密度由高到低依次為皮質、邊緣系統、基底核、視丘、下視丘、中腦、小腦；其中以中腦黑質體的細胞密度為全腦中最高。以腹腔注射酯多醣的方式來誘發周邊發炎發現，微小膠細胞之細胞體會變大，其中以黑質體的變化最為明顯。微小膠細胞的整體面積與密度也發生改變，同樣也是黑質體的變化最為明顯。接著我假設因為周邊發炎後，黑質體的血腦障壁滲漏較多，致使周邊發炎物質進入較多，進而引起微小膠細胞活化較顯著。結果顯示黑質體周圍的血腦障壁滲漏程度在發炎後是全腦中最明顯處。從文獻中得知，周邊發炎因子-腫瘤壞死因子（Tumor necrosis factor receptor, TNFα）能與腦血管內皮細胞上的腫瘤壞死因子接受器（TNFR1）結合，藉以將周邊發炎的訊息傳入大腦，而使得微小膠細胞活化。因此我觀察了發炎後TNFR1在各個腦區的表現程度。結果發現TNFR1在黑質體的表現量較其他腦區來得明顯。又中樞發炎的情況下，活化的微小膠細胞會釋放出一種趨化激素(monocyte chemoattractant protein-1, MCP-1)去召集外來的免疫細胞滲入到腦裡。因此我也觀察了各個腦區MCP-1的表現程度；發現到MCP-1的表現量在每個腦區都有明顯的增加，而黑質體也是表現最多的腦區之一。接著我利用代表單核球細胞與巨噬細胞的標記物-Ly6C去觀察外來的免疫細胞。由結果發現，Ly6C確實在各腦區的表現程度略有不同，特別是在黑質體中可看到有大量的巨噬細胞滲入。總結本研究發現黑質體腦區的微小膠細胞密度為全腦最高，而且於周邊發炎後微小膠細胞活化最顯著。這可能是由於黑質體腦區的TNFR1增加量，於發炎後較其他腦區來得明顯，導致血腦障壁滲漏程度較大，微小膠細胞活化較顯著，以及吸引較多周邊的免疫細胞進入此腦區。因此，黑質體是一個最容易遭受到周邊發炎所影響的一個腦區。

Microglia, one of three glial cell types in the central nervous system (CNS), play an important role as resident immunocompetent and phagocytic cells in the CNS in the event of injury and disease. Numerous studies indicated that peripheral inflammation may induce CNS inflammation and microglia activation. Activation of microglia is associated with cell transformation to phagocytes, capable of releasing potentially cytotoxic substances such as oxygen radicals, proteases, and proinflammatory cytokines. Recent studies indicated that CNS in?ammation and microglial activation is a common component of the pathogenesis for multiple neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, etc. It has been shown that the densities of microglia are unevenly distributed across different brain regions. However, whether the uneven distribution of microglia has any effect on peripheral inflammation-induced microglia activation remains unknown. The objective of this study is to investigate the temporal and spatial profiles of microglia activation after the peripheral inflammation in male C57BL/6 mice. Microglia morphological changes and densities of microglial cells in various brain regions were visualized using an immunohistochemical method with a polyclonal antibody recognizing the mouse ionized calcium binding adaptor protein-1 (Iba-1). Our results showed that substantia nigra (SN) had the highest microglia densities among the brain regions, followed by (in a high to low order) cortex, limbic system, basal nucleus, midbrain, thalamus, hypothalamus, brain stem, and the lowest in cerebellum. Microglia activation (soma enlargement, Iba-1-positive cell area and cell density) were observed after intraperitoneal LPS injection, with the highest degree of change in SN. I hypothesize that the integrity of blood-brain barrier (BBB) is involved in the transmission of inflammatory responses from the periphery to the CNS. The results showed that the BBB leakage was most pronounced in the SN among other brain regions. . Previously, it has been shown that TNFα/TNFα receptor 1 (TNFR1) play critical role in transmitting the inflammation signal from periphery into CNS. Therefore, we also investigated the expression of TNFR1 one day after LPS injection. The results showed that the highest LPS-induced TNFR1 levels was in the SN. Furthermore, activated microglia is capable of release cytokines and chemokines; among them monocyte chemoattractant protein (MCP-1) is vital in attracting peripheral monocytes into CNS. Hence, the levels of MCP-1 were measured. The results showed that the expressions of MCP-1 were elevated in all brain regions, with the highest in the SN. Furthermore, we used the Ly6C, a surface marker for monocytes but not microglia, to distinguish peripheral monocytes from residual microglia and found that the number of Ly6C-positve cells was highest in the SN. In conclusion, this study indicates that SN has the highest microglia density in the CNS. Upon peripheral inflammation stimulation, the degree of microglia activation was most prominent in the SN. This phenomenon may be due to the higher TNFR1 expression in the SN after the stimulation of inflammation, leading to larger leakage of BBB, higher expression of MCP-1 and more monocyte infiltration into the SN. Therefore, the SN is the most susceptible brain region in response to systemic immunological insults.

中文摘要 1
英文摘要 3
緒論 11
一、中樞神經系統之發炎反應 11
二、微小膠細胞(Microglia) 12
三、血腦障壁（Blood-brain barrier, BBB） 17
四、酯多醣（lipopolysaccharide, LPS） 18
研究目的 21
實驗設計 22
目標一：觀察大腦裡微小膠細胞的分佈與其活化後型態之改變 22
建立小鼠腦中微小膠細胞之密度分佈 22
比較周邊發炎後腦中微小膠細胞之密度與型態上之變化 22
目標二：探討造成微小膠細胞在不同腦區活化程度不同之原因 23
比較周邊發炎後血腦屏蔽之滲漏程度 23
目標三：探討造成活化後微小膠細胞密度改變之原因 24
比較周邊發炎後微小膠細胞所釋放的趨化激素改變程度 24
計算從周邊血液滲入大腦裡之免疫細胞在各腦區之表現量 24
材料 25
方法 29
一、實驗動物 29
二、伊凡藍染劑 (Evans Blue) 的製備與動物處理 29
三、灌流取腦 30
四、腦組織的抗凍處理 31
五、冷凍切片 31
六、免疫組織染色法(Immunohistochemistry) 32
七、封片 33
八、統計分析 (Statistical analyses) 33
實驗結果 34
一、小鼠腦中微小膠細胞之密度分佈 34
二、周邊發炎後小鼠腦中微小膠細胞之密度與型態上之改變 34
三、周邊發炎後血腦障壁之滲漏性 35
四、周邊發炎後血腦屏蔽上的腫瘤壞死因子接受器在各腦區之表現 36
五、周邊發炎後微小膠細胞所釋放之趨化激素之表現 36
六、周邊發炎後免疫細胞之滲入程度 37
討論 38
結論 43
參考文獻 44
圖與表 48

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