敗血症所引發的多重器官衰竭是造成病人死亡的主原因之一。引發遠端器官衰竭 的主要原因之一目前認為與器官中微血管的内皮細胞受損有關。敗血症所引發的 促發炎反應細胞激素會破壞内皮細胞之von Willebrand factor(vWF)/ADisintegrin And Metalloprotease with Thrombo Spondin(ADAMTS)13凝血功能平衡，促進器官 内的血液凝集造成器官部分因缺乏養分而壞死。而當内皮細胞受到發炎激素刺激 後維持内皮細胞功能相關之angiopoietin/Tie2 ligand-receptor亦會失調，使内皮細 胞無法藉由eNOS產生足夠之NO，使細胞發生細胞程式化凋亡。而維持内皮細胞 間緊密連結之VE-cadherin也會因為angiopoietin/Tie2失衡造成其構造被破壞，當 VE-cadherin構造不完整時，内皮細胞間的通透性增加，使循環血中的體液滲漏 至組織中造成器官水腫。而發炎反應時内皮細胞表現之黏著分子增加，會造成中 性多形核白血球由血液中轉移到組織的數目增加。組織中浸潤的白血球會分泌釋 出自由基與蛋白質分解酶毒殺外來病原菌，此現象亦會造成器官的氧化壓力增加。 骨鑛中的内皮原生細胞(endothelial progenitor cell, EPC)為血管修復過程中非常重 要的細胞體内的修補機轉，敗血症發生時EPC在血液中的數目會增加，但在敗血 症引發多重器官衰竭病人血中則發現EPC比例下降。目前有動物實驗利用給予 EPC移植治療與内皮細胞損傷之相關心血管疾病。結果發現給予EPC移植可以降 低内皮細胞受損，降低心血管疾病的惡化。麩醯胺(glutamine, GLN)是近年來非 常受到重視的特殊營養素，在免疫調節上具有重要的功能，且是一些快速分裂的 細胞能量來源。GLN被認為在創傷或異化性疾病狀態下是一種必需胺基酸。已 有許多研究證實，補充GLN可減輕氧化壓力並減少多重外傷病人之感染率，本 實驗室先前之研究也顯示，GLN添加可改善敗血症引發之肺臟及腎臟器官受損 之狀態。但都只觀察GLN之添加對敗血症發生時免疫系統的調節。故本實驗利用 三年的時間進行系列的研究，第一年利用給予盲腸結紮及穿刺手術 (cecal-ligation-and-puncture ,CLP)引發敗血症之老鼠GLN的補充後，觀察血中EPC 的比例及器官中内皮細胞受損之評估。第二年擬用小鼠骨髓中之幹細胞，經培養 分化成EPC後，觀察給予不同濃度之GLN培養，對其增生及功能評估。為了模擬 臨床使用EPC移植治療内皮細胞受損的治療方式，第三年之動物實驗利用第二年 之結果，選擇培養於適當GLN濃度之EPC，即分化能力及功能表現較佳的EPC， 經由靜脈注射至接受CLP手術之小鼠，並配合上敗血症後給予GLN之靜脈營養支 持，觀察使用EPC移植治療並配合GLN營養支持下，小鼠遠端器官受損之情形。 本研究結果將可作為臨床上敗血症病人使用EPC細胞移植治療及營養支持之重 要參考依據。 Sepsis-induced endothelial cell (EC) injury is known to be one of the main causes that lead to remote organ dysfunction. Overexpression of pro-inflammatory cytokines result from sepsis dysregulate the balance of von Willebrand factor (vWF)/A Disintegrin and Metalloprotease with Thrombo Spondin (ADAMTS)13 system and thus activate capillary platelet aggregation and induce organ ischemia. Besides, the alteration of angiopoietin/Tie2 ligand-receptor system in EC downregulate the activity of endothelial nitric oxide synthase and VE-cadherin expression that may increase endothelial permeability and accelerate the apoptosis of EC. Endothelial progenitor cell (EPC)s are circulating endothelial precursor cells associated with sepsis. Recent study demonstrated that circulating EPCs were increased in septic patients while non-survivors had significantly lower numbers of EPCs than survivors. Animal study showed that facilitate EPC recruitment promote the repair of EC and exogenous EPC administration improve the survival in sepsis. Glutamine (GLN) is a specific nutrient with immunomodulatory properties. GLN is considered as essential amino acid following critical illness and injury. Previous studies showed that GLN attenuated oxidative stress and reduce infectious complications in trauma patients. Studies performed by our laboratory also found that GLN balanced the T cell polarization and alleviate the inflammation and damage of organs induced by sepsis. However, no study investigates the role of GLN on the regulation on EPC in sepsis. Also, the synergistic effect of GLN with different dose of EPC on tissue injury in sepsis remains unknown and require investigation. We plan to carry out this study in 3 consecutive years. In 1st year, mice are subjected to cecal ligation and puncture (CLP) and GLN will be administered to evaluate the expression of blood EPC in association with the EC function, adhesion molecule expressions and tissue injury. In the 2nd year, stem cells obtained from mice bone marrow will be cultured and differentiate to EPC. Different dosages of GLN will be cultured with EPC to evaluate the rd optimal condition for EPC proliferation and function. In the 3 year, the optimal conditioned EPC accompanied with GLN will be administered to CLP mice to evaluate whether exogenously EPC and GLN may have synergistic effects in improving EC function and thus attenuating organ injury in sepsis. This study will be the first study to investigate the effect of GLN on EPC regulation, and the results may provide useful information in clinical application in nutrition support and medical therapy in septic condition.
