臺灣博碩士論文加值系統

在阻塞型先天性心臟疾病中，可透過注射前列腺素而維持動脈導管的暢通來拯救其性命，但也伴隨著許多藥物副作用。因此發現一個有效且安全的藥物，用以維持動脈導管暢通在兒童加護照顧中是很重要但卻缺乏的課題。動脈導管關閉包含兩個階段：血管收縮與重塑。我們假設可溶性鳥苷酸環化酶活化劑（soluble guanylyl cyclase (sGC) activator）cinaciguat透過sGC-PKG訊息使得動脈導管舒張及抗重塑來維持其暢通。在活體內實驗，我們逐一檢查cinaciguat（10 mg/kg腹內注射）作用在初生鼠出生第0及第2小時，在動脈導管型態及組織學上所表現出的效果。動脈導管的管徑通暢程度及重塑狀況將被檢視。在活體外實驗，初生鼠的動脈導管被分離出來，用以檢查cinaciguat對其血管張力的影響。在體外實驗，由初生鼠所分離培養的動脈導管平滑肌細胞被用以檢視抗重塑的機制。
結果顯示，初生鼠在出生2小時其動脈導管幾乎自行關閉，而在出生0小時使用cinaciguat可以預防動脈導管關閉，其動脈導管/主肺動脈外徑比值（DA/PA ratio）比控制組增加14.3%且空腔占比增加64.4%（p < 0.05)。在活體外實驗，cinaciguat（10-9~10-7 M）顯著地減弱氧氣所誘導的動脈導管收縮且有劑量依賴性效果。在體外實驗，cinaciguat抑制angiotensin II所誘發的動脈導管平滑肌增生及遷移。Cinaciguat抑制angiotensin II所誘發的ERK、JNK、p38及Akt訊息活化。總結，cinaciguat藉由血管擴張及抗重塑來防止出生後動脈導管的關閉。其中血管擴張及抗重塑的機制包含透過PKG訊號來減弱ERK、JNK、p38及Akt達到抗增生及抗遷移。因此sGC-PKG訊息路徑有希望成為臨床處置動脈導管上的治療標的。

Ductus arteriosus (DA) patency can be life-saving in some obstructive congenital heart lesions and is achieved by infusion of prostaglandin E1, which unfortunately has lots of side effects. Therefore, an effective and safe agent maintaining DA patency is important in pediatric intensive care but is lacking. DA closure consists of two phases: vasoconstriction and remodeling. We hypothesized that cinaciguat, a soluble guanylyl cyclase (sGC) activator, can maintain DA patency by vasodilatation and anti-remodeling through sGC-PKG signaling. In vivo, we serially examined effects of cinaciguat (10 mg/kg, ip) on DA morphology and histology in neonatal rats at 0 h and 2 h after birth. The degree of DA patency and remodeling were examined. Ex vivo, neonatal DA rings were used to examine effects of cinaciguat on vascular tone. In vitro, DA smooth muscle cells (DASMCs) cultured from neonatal rats were used to investigate mechanisms of anti-remodeling.
We found that in control rats at 2h after birth, DA almost spontaneously closed. Cinaciguat at birth prevented DA closure with an increased DA/PA external diameter ratio by 14.3 % and an increased luminal patency by 64.4% than control at 2h. In addition, cinaciguat inhibited DA remodeling with a reduced intima/media diameter ratio of 21.9 % than control (p<0.05). Ex vivo, cinaciguat significantly attenuated oxygen-induced DA ring vasoconstriction in a dose-dependent manner (10-9~10-7 M). In vitro, cinaciguat inhibited antiotensin II (Ang II) -induced DASMC proliferation, migration. Finally, cinaciguat inhibited Ang II-induced ERK, JNK, p38 and Akt activation. In conclusion, cinaciguat prevents postnatal DA closure by both vasodilation and anti-remodeling. The mechanisms underlying vasodilatory and anti-remodeling effects consist of anti-proliferation and anti-migration, with attenuation of ERK, JNK, p38 and Akt signaling, through the PKG pathway. Therefore, sGC/PKG pathway can be a promising therapeutic target for clinical management of ductus arteriosus.

中文摘要 1
Abstract 3
縮寫表 4
第一章　緒論 6
動脈導管簡介 6
動脈導管在臨床上的角色 7
動脈導管的藥物選擇及其副作用 7
動脈導管關閉的機轉 8
目前臨床藥物的機轉 12
利鈉胜肽在心血管的角色 13
Angiotensin II對心血管的影響 15
Cinaciguat在心血管的角色 17
第二章　研究目的 20
第三章　研究材料 22
實驗動物 22
實驗細胞 22
實驗用藥 22
實驗溶液 22
實驗儀器 22
電腦軟體 23
動脈導管平滑肌細胞培養 23
細胞存活檢測：MTT assay 24
西方點墨法（Western blotting） 24
第四章　實驗方法 25
動物實驗 25
動脈導管血管張力實驗 27
動脈導管平滑肌細胞實驗 28
統計方法 35
第五章 實驗結果 36
Cinaciguat維持初生鼠動脈導管的通暢 36
Cinaciguat能使動脈導管血管擴張且有劑量依賴性效果 38
PKG inhibitor減弱cinaciguat的血管擴張效果 38
Cinaciguat抑制動脈導管平滑肌細胞的增生及遷移 38
Cinaciguat減少ERK、JNK、p38及Akt的磷酸化 39
第六章 討論 40
Cinaciguat對初生鼠動脈導管有抗重塑的作用 40
Cinaciguat藉由PKG訊息使得動脈導管劑量依賴性地血管擴張 42
Cinaciguat抑制動脈導管平滑肌細胞的增生及遷移 44
Cinaciguat抑制MAPK及Akt的細胞訊息 45
結論 46
參考文獻 47
第六章 附圖 52
學術研討會及論文發表 72

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