臺灣博碩士論文加值系統

摘要

嚴重敗血症患者呈現全身性炎症反應、促凝血素質特異體質（Prothrombotic diathesis)和纖維蛋白溶解失調三種現象。近年來研究指出活性蛋白質C（Activated protein C；APC）可藉由抑制血栓及防止多重器官衰竭的發生來降低敗血症病患20 %的死亡率；然而APC如何抑制細菌內毒素（Lipopolysaccharide；LPS）對內皮細胞傷害的機制目前尚不清楚。在此我們首先利用細胞存活率來描述細胞在不同生長狀態時LPS對細胞死亡的影響，結果顯示未長滿及長滿後1和3天的細胞其存活率在加入LPS後迅速的降低，接著才又短暫的回升，而剛長滿的細胞則因對LPS較為敏感而持續降低其存活率。當加入APC作用時， LPS的持續存在會減少APC對細胞存活率提升的能力，此外APC能藉由維持eNOS蛋白在細胞內的含量來抑制LPS對細胞所造成的死亡，尤其以剛長滿之細胞最為明顯。另一方面APC也能夠藉由增加細胞質內β-catenin的降解以及藉由清除細胞核中的β-catenin來抑制細胞進入S phase來提升LPS處理後細胞的存活率。此外Wnt agonist確實有抑制APC降解細胞質內β-catenin的能力造成最終細胞存活率的下降。

Abstract
The patient with severe sepsis appears inflammation, prothrombotic diathesis, and impairment of fibrinolysis in the whole body. Although the studies show that activated protein C (APC) is effective to reduce mortality by nearly 20% in sepsis patients via inhibiting thrombosis and preventing multiple organ dysfuction, the underlying mechanism of APC on the survival of lipopolysaccharide (LPS)-activated endothelial cells (ECs) remains unclear. Here, we first characterized cell viability after LPS challenge using different growth states of ECs. The results show that the cell viability is quickly decreased after LPS treatment and then is transiently increased with time in the subconfluent cells and the cells of 1 and 3 days after confluence. In the recently confluent cells, the cell viability decreases monotonously after LPS treatment. During the APC treatment, the presence of LPS reduces the effect of APC on cell viability. Furthermore, APC is able to reverse LPS-modulated cell death and downregulation of endothelical nitric oxide synthase (eNOS) protein, especially in the cells of recent confluence. On the other hand, APC may increase the cell viability of LPS-treated ECs through increasing the degradation rate of cytosolic β-catenin and scavenging nuclear β-catenin by binding with the APC-induced genes to prevent cell entry into S phase. In addition, the Wnt agonist is able to inhibit the action of APC on degradation of cytosolic β-catenin and cell viability.

目錄

頁次
中文摘要…………………………….……………………………….....Ⅰ
英文摘要…………………………….……………………………….....Ⅱ
目錄……………………………….……………………………….........Ⅳ
圖目錄……………………………….……………………………….....Ⅷ
表目錄………………………………..…………………….……...……Ⅹ
符號…………………………………..………………….….........……..XI
第一章 緒論………….………………………….………………...…...1
1-1 前言………………………………………….…………………..1
1-2 文獻回顧……………………………………………………..….2
1-2.1 血管內皮細胞 ……………..…………..………………....2
1-2.2 一氧化氮與一氧化氮合成酶…………..……………...….3
1-2.3 一氧化氮與細胞凋零的關係…………..……….……...…5
1-2.4 細胞凋亡的指標Caspase 3……………………....….….…6
1-2.5 加護病房中主要的死因-敗血症……………………..…...9
1-2.6 敗血症的相關因子-細菌內毒素…………...…..………..13
1-2.7 治療敗血症的新藥-活性蛋白質C…………………..….15
1-2.8 APC的訊號傳遞接受器Proteinase-activated receptor
1……………………………………………………….….17
1-2.9 與細胞增生有關的因子β-catenin…………………..…..18
1-2.10人類疾病中與細胞弁鉬棆爬傢鰝熙J白CBP/ p300..………………………………………………….….20
1-3 研究動機與目的……………………………………………….21
第二章 實驗藥品、儀器與器材……………………….……..….…..23
2-1 實驗藥品…………………………………………………….…23
2-1.1 細胞培養…………………..………………………………23
2-1.2 Lysis buffer contents……………..………………………...24
2-1.3 蛋白質濃度測定…………………………………………..24
2-1.4 Caspase 3活性測量……………………….……..……...…24
2-1.5 NO測量…..……………..………………………………….25
2-1.6 Cell viability……………………..…………………………25
2-1.7 Western blotting………...…………………………………..26
2-2 實驗儀器…………………………………………………….....27
2-3 實驗器材……………………………………………..….……..29
第三章 實驗步驟與原理………………………………..…….……...31
3-1 人類大主動脈血管內皮細胞的培養……………..….………..31
3-2 蛋白質濃度測定………………………...…….……………….34
3-3 Caspase 3活性的測量…..…..………...……………………….35
3-4 一氧化氮定量……………….…………………………………38
3-5 Cell viability………………………….………………………...40
3-6 Preparation of nuclear extract-freeze and thaw….……………..41
3-7 Preparation of nuclear extract－Dounce……………………….43
3-8 Western blotting………….…………………………………….44
3-9 Data analysis…………………………………………………...49
第四章 實驗結果與討論……………………………………..…....…50
4-1 LPS及APC對內皮細胞存活率的影響…………………..…...50
4-1.1 LPS對不同生長狀態下內皮細胞存活比例隨時間之變化……………..………………………………………...….50
4-1.2 APC對不同生長狀態的內皮細胞在LPS不同處理時間下細胞存活比例之變化……..……………..………………..52
4-2 APC對不同生長狀態下的內皮細胞在LPS不同處理時間所引起之eNOS降解之抑制………………………...……………..52
4-3 APC藉由增快β-catenin降解抑制LPS所引起造成內皮細胞的死亡……………………………………………………………54
4-3.1不同生長天數的內皮細胞對β-catenin蛋白質之表現……………………………………………………..……54
4-3.2 APC對LPS處理之細胞的β-catenin降解之影響…………………………..……..………………………..58
4-3.3 wnt agonist對細胞存活及β-catenin降解之影響…………………..……………………...…………….....62
第五章 結論未來展望………………………………….………….....65
5.1 結論………………….………………………….….……….….65
5.2 未來展望………………………....……………..….…………..65
附錄A……………………………………………………………...……67
參考文獻………………….……………………………………….……69









圖目錄

頁次
圖1-2.1 APC活化PAR 1的機制………………………………..……....2
圖1-2.2 一氧化氮生成示意圖………………………………….………5
圖1-2.3 一氧化氮致使細胞凋亡的機制…………………………….…6
圖1-2.4 敗血症的機制……………………………………………..….11
圖1-2.5 細菌細胞壁的結構……………………………………...…....12
圖1-2.6 內毒素引發敗血症的機轉…………………………………...13
圖1-2.7 LPS結構圖……………...……………………………………..14
圖1-2.8 內毒素造成細胞凋亡的訊號傳遞…………………….….….16
圖1-2.9 PAR的活化機制……………………………………………....18
圖1-2.10 β-catenin的活化機制………………...…............................…20
圖1-2.11 CBP與p300蛋白的比較………………………………….…21
圖4-1.1 LPS對不同生長狀態下內皮細胞存活比例隨時間之變化….51
圖4-1.2 APC對不同生長狀態的內皮細胞在LPS不同處理時間下細胞存活比例之變化……....………………..……………………..53
圖4-2.1 APC對不同生長狀態下的內皮細胞在LPS不同處理時間所引起之eNOS降解之變化……………..…………………...…....55
圖4-2.2 APC對不同生長狀態下的內皮細胞在LPS持續處理所引起之內皮一氧化氮合成酶降解之變化…………..……………..…57
圖4-3.1 β-catenin在不同生長狀態下內皮細胞的表現差異………….59
圖4-3.2 β-catenin在不同生長狀態下內皮細胞的表現差異……….....60
圖4-3.3在細胞核與細胞質分離下不同APC及LPS處理時間對β-catenin降解之比較………………………..………………...61
圖4-3.4 不同狀態下wnt agonist對細胞存活比率之影響……………63
圖4-3.5 不同狀態下wnt agonist對β-catenin表現量之影響…………64
圖5-1.1 APC降解β-catenin致使細胞存活的路徑圖…………………66
圖A-1 APC及LPS在不同狀態下對Caspase 3活化的影響………….67












表目錄

頁次
表1-2.1 Caspase的分類……………………………………………...…..8
表1-2.2臨床上敗血症及相關症狀的定義…………………………….10
表4-1.1 APC抑制LPS作用的比較……………………………………54


























符號

CrmA: Cytokine response modifier A
ERICE: Evolutionary related ICE
FLICE: Fas-associated death domain-like IL-1β-converting enzyme
IAP: Inhibitors of apoptosis
ICAD: Inhibitor of caspase-activated DNAase
ICE: Interleukin-1β converting enzyme
MICE: Mini ICE
PARP: Poly(ADP-ribose)polymerase-1
PKC: Protein kinase C
SREBP: Sterol regulatory element-binding protein

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