系統識別號 U0026-3005201210395300
論文名稱(中文) 過量麻醉劑異丙酚導致體外內皮細胞毒性與體內內皮障壁功能失調
論文名稱(英文) Anesthetic Propofol Overdose Causes Endothelial Cytotoxicity In Vitro and Endothelial Barrier Dysfunction In Vivo
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 100
學期 2
出版年 101
研究生(中文) 林明忠
研究生(英文) Ming-Chung Lin
學號 S97971159
學位類別 碩士
語文別 英文
論文頁數 37頁
口試委員 指導教授-林秋烽
中文關鍵字 異丙酚  內皮細胞  肝醣合成酶激酶  細胞凋亡  細胞壞死  血管通透性 
英文關鍵字 propofol  endothelial cells  GSK-3  apoptosis  necrosis  vascular permeability 
中文摘要 異丙酚 (Propofol),是一個短效的靜脈麻醉劑。臨床上如果過量及過長時間使用異丙酚可能會造成多重器官/組織之細胞毒性,包括了腦、心臟、腎臟、骨骼肌細胞與免疫細胞等;然而其確切的致病機轉至今仍未被報導,特別是在血管內皮細胞的部分。根據我們先前的研究成果顯示巨噬細胞在過量的異丙酚使用下,其肝醣合成酶激酶 (GSK)-3會活化而促進細胞凋亡。由於異丙酚是使用在全身血液循環中,因此我們推測過量之異丙酚會透過活化血管內皮細胞之GSK-3進而造成細胞毒殺作用。本研究之結果顯示過量的異丙酚會抑制人類微血管內皮細胞與人類主動脈內皮細胞之生長。在投予藥物後,大多數內皮細胞會進行與凋亡蛋白酶(caspase)無關之類壞死狀態的細胞死亡;溶酶體的膜通透後續造成組織蛋白酶 (cathepsin) D的活化而決定了類壞死狀態的細胞死亡;且此細胞死亡無關乎自體吞噬作用。再者,過量的異丙酚也會導致caspase依賴性之細胞凋亡;內皮細胞之caspase-3活化後會扮演粒線體膜電位喪失後之下游角色;然而不管是溶酶體之cathepsin B或是cathepsin D,對於此細胞凋亡的形成都是非必要的。且顯而易見地,GSK-3的活化對於過量之異丙酚所導致的細胞凋亡是必要的,但無關乎其所引起之類壞死狀態的細胞死亡。此外,為小鼠腹腔注射過量的異丙酚會導致腹膜血管通透性增加。總結,這些研究結果展示了過量異丙酚在體外對於內皮細胞的細胞毒殺作用,包括了cathepsin D所調控的類壞死狀態之細胞死亡和GSK-3所調控之細胞凋亡,與異丙酚在體內所導致的血管內皮障蔽之功能失調。
英文摘要 An overdose of propofol following a prolonged treatment may cause cellular cytotoxicity in multiple organs/tissues, such as brain, heart, kidney, skeletal muscle, and immune cells; however, the molecular mechanism remains undocumented particularly in vascular endothelial cells. Our previous studies showed that activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes under overdose propofol treatment. Regarding the medication of propofol in circulation, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity through GSK-3. Propofol overdose (100 µg/ml) induced growth inhibition in human aortic endothelial cells and human microvascular endothelial cells. After treatment, most of endothelial cells underwent caspase-independent necrosis-like cell death. Activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death independent of autophagy. Furthermore, propofol overdose also induced caspase-dependent apoptosis. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, neither lysosomal cathepsin B nor cathepsin D was required for apoptosis. Notably, activation of GSK-3 was essential for overdose propofol-induced apoptosis but not necrosis-like cell death. Intraperitoneal treatment of overdose propofol in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated apoptosis, on endothelial cells in vitro, and the endothelial barrier dysfunction by propofol in vivo.
論文目次 中文摘要 I
Abstract II
誌謝 III
Abbreviations IV
Contents V
List of Figures VII
1.1 Introduction of Propofol 1
1.2 Pathogenesis of Propofol Infusion Syndrome 1
1.3 Propofol-induced Apoptotic Cell Death 2
1.4 Propofol-induced Necrotic Cell Death 2
3.1 Reagents and Antibodies 5
3.2 Cell Cultures 5
3.3 Animals 6
3.4 Permeability Changes 6
3.5 Viability Assay 7
3.6 Cytotoxicity Assay 7
3.7 Apoptosis Assay 8
3.8 LMP Assay 8
3.9 Mitochondrial Functional Assay 9
3.10 Western Blotting 9
3.11 Lentiviral-based Short Hairpin RNA Transfection 10
3.12 Statistical Analysis 11
4.1 Propofol Overdose Induced Growth Inhibition, Apoptosis, and Caspase- independent Necrosis-like Cell Death in Human Endothelial Cells 12
4.2 Propofol Overdose Induced LMP and Lysosomal Protease Cathepsin D- regulated Necrosis-like Cell Death Independent of Autophagy 13
4.3 Propofol Overdose Induced Caspase-independent MTP Loss and Caspase-3-dependent Cell Apoptosis 14
4.4 GSK-3 Activation, GSK-3-regulated Mitochondrial Apoptosis, and GSK-3-independent Necrosis-like Cell Death were Involved in Propofol Overdose-induced Cytotoxicity 15
4.5 Propofol Overdose Increases Peritoneal Vascular Permeability in BALB/c Mice 16
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