臺灣博碩士論文加值系統

敗血症的定義為感染所引發之全身性發炎反應，晚期很容易產生敗血性休克，病人會出現嚴重低血壓及對升壓劑產生血管低反應性的情形。由於血壓無法回復，導致身體組織與器官持續處於低灌流狀態，最後則會導致多重器官功能異常而死亡。因此，研究並了解敗血性休克時血管低反應性的機轉是目前非常重要的課題。
近年來許多學者針對鈣離子致敏化機轉來研究血管平滑肌的收縮，其中，此機轉中的小 G 蛋白 RhoA 對於血管收縮的調控扮演著重要角色。RhoA 和其下游 Rho-kinase 的活化會使肌凝蛋白輕鏈去磷酸酶失去活性，造成血管的收縮。 RhoA 的活性會受到 GEF (guanine nucleotide exchange factor)、 GAP (GTPase activating protein) 和 GDI (GDP dissociation inhibitor) 三種蛋白質調控，因此可藉由影響這三種蛋白質去調控 RhoA 的活性。研究指出， Src 家族酪胺酸蛋白激酶 (Src family tyrosine kinase, SFK) 具有活化 GEF 的作用，因此對 RhoA 的活化也扮演著重要角色。另外，也有研究指出在鈣離子非依賴性血管收縮方面，蛋白質激酶 C (protein kinase C, PKC) 產生的血管收縮是經由活化 RhoA 所造成的。本實驗的目的主要探討，在敗血性休克老鼠的胸主動脈中，RhoA 活性的變化，而 PKC 和 SFK 對於 RhoA 活性的調控扮演的角色。
本實驗以靜脈注射內毒素的方式誘發老鼠產生敗血性休克，再將老鼠犧牲並取出其胸主動脈，觀察血管 RhoA表現與活性的變化，並評估 PKC 和 SFK 對 RhoA 活性的調控。結果發現在內毒素血症老鼠主動脈中，RhoA 的活性顯著下降。給予 PKC 和 SFK 抑制劑 (分別為 GF-109203X 和 PP2) 後，對敗血性休克老鼠血管的 RhoA 活性產生抑制作用。 GF-109203X 在敗血性休克的情況下，對 SFK 的活性產生明顯的抑制作用。換言之，在敗血性休克下， PKC 和 SFK 對 RhoA 活性有明顯的調控作用； PKC 對於 SFK 亦有明顯的活化作用。
綜合實驗結果顯示， lipopolysaccharide (LPS) 誘發老鼠內毒素敗血性休克其胸主動脈產生血管低反應可能與 RhoA 活性降低密切相關，且 PKC 和 SFK 對 RhoA 活性之調控扮演重要角色。另外， PKC 亦可透過 SFK 去調控 RhoA 活性。

Sepsis is the systemic inflammatory response with infection. Usually, septic shock is occurred at the late phase of sepsis. Septic shock is characterized by severe hypotension and reduced response to vasopressor agents, called vascular hyporeactivity. The hypotension and vascular hyporeactvity are associated with the development of multiple organs dysfunction which causes death eventually. Thus, it is important to investigate the mechanism of vascular hyporeactivity in septic shock.
Among the cell signaling pathways that are crucial to control vascular tone, Ca2+-sensitization contraction has become more and more important. The small GTP-binding protein, RhoA, plays a crucial role in mediating smooth muscle contraction. Activation of RhoA leads to inactivation of myosin light chain phosphatase via activation of Rho-kinase. The activation of RhoA is controlled by three mediators, GEF (guanine nucleotide exchange factor), GAP (GTPase activating protein) and GDI (GDP dissociation inhibitor). Among these mediators, GEF has been regarded as the most important one for RhoA activation. It has been shown that Src family tyrosine kinase (SFK) can activate RhoA via phosphorylation of GEF. In addition, it is reported that activation of PKC can lead to Ca2+-independent vasocontraction through activation of RhoA. The purpose of this study was to investigate the role of PKC and SFK in RhoA activity in thoracic aortae from endotoxemic rats.
Rats received an intravenous injection of lipopolysaccharide (LPS, 10 mg/kg) for 4 hours. After then, rats were sacrificed and the thoracic aortae were excised and immediately incubated in GF-109203X (PKC inhibitor) and PP2 (SFK inhibitor). We found that RhoA activity was decreased significantly in aortae from endotoxic rats. GF-109203X and PP2 had inhibitory effect in aortae from endotoxic rats only. In addition, the phosphorylation of SFK was decreased by GF-109203X in endotoxemic rats.
In conclusion, PKC and SFK might play a more crucial role in RhoA activation and PKC might play a more important role in activation of SFK endotoxemic rats. These results suggest that RhoA is related to vascular hyporeactivity caused by LPS-induced septic shock. In addition, PKC and SFK might be targets for improving vascular hyporeactivity.

目錄 I
圖目錄 II
英文縮寫對照一覽表 IV
中文摘要 V
ABSTRACT VII
第一章 緒論 1
第二章 材料與方法 14
第三章 實驗結果 27
第四章 討論 33
第五章 結論 45
參考文獻 63

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