臺灣博碩士論文加值系統

目前文獻對中樞神經系統感染克雷白氏肺炎桿菌後，宿主的免疫和發炎反應了解仍然有限。本實驗室之前已建立一個克雷白氏桿菌感染腦膜腦炎的大鼠動物模式，我們發現雖然腦脊髓液中白血球數目增加，但其數量不足以解釋腦脊髓液中細胞素濃度遠高於血清中濃度的現象；而且膠質細胞(主要是星狀膠質細胞和微膠質細胞)之數種細胞激素mRNA與蛋白質表現之時間點亦較腦脊髓液白血球增加更早。在本篇論文裡，我們於是提出以下假說：膠質細胞引起之發炎反應和氧化壓力，是造成克雷白氏肺炎腦膜腦炎的神經元死亡和伴隨的後遺症之重要因素；而黃芩素（baicalein），一種黃酮類化合物，因具有抗發炎和抗氧化的作用，故可以保護克雷白氏肺炎腦膜腦炎的神經元傷害。本論文之動機即是研究驗證此假說之正確性。
於本論文的第一部分，我們利用初代培養之Sprague-Dawley大鼠大腦皮質細胞研究黃芩素對克雷白氏桿菌感染之大鼠大腦皮質細胞之影響。免疫細胞化學染色及流式細胞儀分析顯示我們之初代培養之大腦皮質細胞中大約含49%的神經元，38%的星狀細胞和10%的微膠質細胞組成。感染克雷白氏肺炎桿菌後，細胞存活率隨時間增長而下降；同時給予黃芩素(10, 30, 50, 100M)可以增加細胞存活率。流式細胞儀分析顯示，克雷白氏肺炎桿菌造成神經元和微膠質細胞死亡，但不造成星狀細胞死亡。給予黃芩素不僅可以減弱克雷白氏肺炎桿菌引起的細胞死亡，也可以使星狀細胞增殖。黃芩素(0.3- 100M) 降低細胞培養液中發炎介質如NO、TNF-、IL-1、IL-6等的濃度，且黃芩素(10, 30, 50, 100M)此作用與濃度相關，黃芩素(0.3, 30, 50,M)亦可以降低細胞內氧化活性物質(ROS)的產生。克雷白氏肺炎桿菌之感染亦會使初代培養之大腦皮質細胞隨時間增加誘導與發炎相關蛋白質之mRNA表現，例如inducible NOS (iNOS)，cyclooxygenase-2 (COX-2)，heme oxygenase-1 (HO-1)和趨化素 macrophage inflammatory protein-2 (MIP-2)；而給予黃芩素均可以減少這些發炎相關蛋白質mRNA之表現量。
在本篇論文的第二部分，我們利用先前已建立的腦室接種克雷白氏肺炎桿菌的大鼠活體動物模式，來研究黃芩素的影響。在接種克雷白氏肺炎桿菌後，給予黃芩素(30 mg/kg , i.p.) ，可以減少腦脊髓液和血中白血球數及降低腦組織中致炎性細胞素（包括腫瘤壞死因子-、介白質-1、介白質-6）的濃度。
綜括而言，我們的結果顯示黃芩素對克雷白氏肺炎桿菌感染引起之腦膜腦炎具有抑制膠質細胞所引起之發炎反應及降低細胞內活性氧物質生成之作用。所以，我們認為黃芩素在治療克雷白氏肺炎桿菌感染所引起之腦膜腦炎，或許可以作為一個有效之輔助性藥物，以提供有益療效。

Klebsiella pneumoniae (K. pneumoniae) is one of the most common causative pathogens of community- and nosocomial-acquired bacterial Klebsiella pneumoniae in Taiwan. Despite great advances in antimicrobial therapy and supportive care, the fatality of K. pneumoniae meningitis remains high at 30-40%, and survivors suffer from long-term severe neurological sequelae. However, the host immune/inflammatory response following CNS infection by K. pneumoniae remains poorly understood. We have previously established a rat model of K. pneumoniae meningoencephalitis and found that leukocytosis could not account for the much higher levels of cytokines in cerebrospinal fluid than in serum. Moreover, the rapid increases of these cytokines at the mRNA and protein levels in glia cells (astrocytes and microglia) occur earlier than the onset of leukocytosis.
In the first part of this thesis we examined the effects of baicalein, a favonoid in vitro using primary cortical cultures prepared from neonatal Sprague-Dawley rats. Immunocytochemical staining and flow cytometric analysis indicated that our primary cortical cultures consisted of 49% neurons, 38% astrocytes and 10% microglia. Exposure to K. pneumoniae resulted in a time-dependent reduction in cell viability and co-treatment with baicalein improved cell survival in a concentration-dependent manner. Flow cytometric analysis further indicated that exposure to K. pneumoniae resulted in cell death of neurons and microglia bur not astrocytes. Baicalein (10, 30, 50, 100 M) not only attenuated K. pneumoniae- incuced cell death but also resulted in proliferation of astrocytes. Baicalein(10, 30, 50, 100 M) also concentration- dependently reduced levels of inflammatory mediators such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-), interleukin (IL)-1 and interleukin (IL)-6 in culture media as well as intracellular ROS production in primary culture exposed to K. pneumoniae (1.5 X 106 CFU) for 24 hr. Exposure to K. pneumoniae also time-dependently upregulated in mRNA levels of inducible NOS (iNOS), cyclooxygenase-2 (COX-2), heme oxygenase-1 (HO-1) and a chemokine macrophage inflammatory protein-2 (MIP-2) and baicalein treatment attenuated the upregulation on the mRNA expression of these inflammation-related proteins.
In the second part of this thesis, we examined the effect of baicalein in our previously developed animal model. Baiclein (30 mg/kg i.p.) administered after i.c.v. K. pneumoniae inoculatin decreased leukocyte counts in CSF and blood, and reduced the cytokines (TNF-, IL-1 and IL-6) levels of brain tissue. Taken together, our data suggest that baicalein has anti-inflammatory and anti-oxidant effects in K. pneumoniae meningoencephalitis and therefore has a potential to be used as a supportive therapy in meningoencephalitis.

中文摘要
英文摘要
第一章 緒論
第二章 實驗目的
第三章 材料與方法
第四章 實驗結果
第五章 討論
第六章 結論
圖表次
參考文獻

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