活性蛋白質C/TCF DNA誘餌對細菌內毒素處理之人類主動脈內皮細胞中β-catenin含量及分佈的影響

敗血症患者會呈現全身性發炎、低血壓、凝血功能失衡及器官官能障礙，在臨床研究上，活性蛋白質C已被證實可將嚴重敗血症病患的死亡率由30.8%降低至24.7%，但目前對於活性蛋白質C對受內毒素感染的血管內皮細胞影響的機制尚未完全清楚。由實驗室先前結果得知，活性蛋白質C 可以有效的降低受內毒素感染的人類主動脈內皮細胞中的β-catenin含量並減少內皮細胞的死亡，所以我們推測細胞的凋亡與細胞中β-catenin含量的降低有相當的關係。為了觀察主動脈細胞受到內毒素感染後，細胞內β-catenin含量的變化，實驗建構pEGFP-β-catenin質體，進而表現EGFP及β-catenin的融合蛋白，之後將此質體經由轉染(transfection)的方式轉染至主動脈內皮細胞中，再利用螢光顯微鏡觀察內皮細胞受內毒素感染後，以及感染後分別進行基因以及藥物治療，β-catenin在內皮細胞中的動態變化。結果發現內皮細胞以內毒素處理一小時後，細胞的螢光亮度沒有明顯的變化；以內毒素對內皮細胞前處理六小時後，加入APC以及TCF DNA Decoy的組別，細胞的亮度有隨時間遞減的趨勢。顯示APC及TCF假目標DNA片段可以有效降低細胞質中β-catenin含量。

關鍵字

活性蛋白質C ； TCF DNA誘餌；細菌內毒素；人類主動脈內皮細胞

並列摘要

The patient with severe sepsis appears systemic inflammatory reaction, hypotension, prothrombotic diathesis, and organ dysfunction. In the previous study, activated protein C (APC) is effective to reduce the mortality from 30.8% to 24.7% in sepsis patients, However, the underlying mechanism of APC on the survival of lipopolysaccharide-activated endothelial cells (ECs) remains unclear. In our previous study, we found that APC is able to reduce the level of cytosolic β-catenin, so we further hypothesized that the effect of APC on cell survival and proinflammatory cytokine production may be due to inhibiting the β-catenin-TCF-mediated gene expression. To observe the dynamic concentration change of β-catenin in human aortic endothelial cells(HAECs) in response to lipopolysaccharide, APC, and DNA decoy, we transfected HAECs with a plasmid pEGFP-β-catenin, expressing β-catenin fusion protein with enhanced green fluorescent protein (EGFP). We observed that the fluorescence intensity in the cytosol showed insignificant change after HAECs treated with 0.1 g/mL lipopolysaccharide for one hour. For cells pretreated with lipopolysaccharide for six hours, the addition of APC or TCF DNA Decoy, decreased the cytosolic fluorescence intensity. These results suggest the efficacy of APC and TCF DNA decoy in reducing the amount of cytossolic β-catenin in HAECs.

並列關鍵字

β-catenin ； Lipoplysaccharide ； Activated Protein C ； TCF DNA decoy

參考文獻

24. Peng-Chan Lin, J.-C.L., Molecular Biology and Signal Pathway of Colorectal Cancer. 中華癌醫會誌 (J. Chinese Oncol. Soc.) 2008: p. 157-166.

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活性蛋白質C/TCF DNA誘餌對細菌內毒素處理之人類主動脈內皮細胞中β-catenin含量及分佈的影響

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