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研究生(外文):Shyh-Jou Shieh
論文名稱(外文):From the Anatomic and Clinical Studies of Microsurgical Perforator Flap Transplantation to the Development of an in Vivo Platelet-Rich Arterial Thrombolytic Model
指導教授(外文):Hua-Lin WuHaw-Yen Chiu
中文關鍵詞:穿通枝皮瓣前外股皮瓣動脈血栓活體模型富含血小板動脈血栓血流動態即時偵測nitric oxide synthase缺氧再回氧損傷缺血再灌流損傷
外文關鍵詞:perforator flapanterolateral thigh flapin vivo arterial thrombosis modelplatelet-Rich arterial thrombosisdynamic real-time measurement一氧化氮合成脢hypoxia-reoxygenation injuryischemia-reperfusion injury
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血管吻合後如發生血栓,會導致皮瓣壞死。許多溶解血栓製劑在臨床上被用於搶救瀕臨壞死的皮瓣,全身性出血及再栓塞是這些溶解血栓製劑最大的問題。由文獻上已知顯微血管吻合後的血栓主要成分是血小板,傳統的溶解血栓製劑,如tissue-type plasminogen activator,streptokinase,urokinase等,各有其優、缺點,希望實驗室基因改造重組的鏈激?,有更好的穩定性及對血小板血栓的專一性。在試管內的實驗顯示,突變種鏈激脢SK(16-378)-K59AK332A有較好的穩定性,融合蛋白SK(16-378)-K59AK332A-γ-(Gly)7-RGDS對血小板結合力較高,但在複雜的活體,則有賴可重覆性的動物血栓模型來測試。本實驗建立一可重覆性較高之動物活體血栓模型,並已完成初步之基因重組蛋白之活體測試。
組織血液循環再重建後,可能引發細胞的缺血再灌流損傷,導致局部組織的纖維化或全身性的症狀。本實驗在試管內(rat VSM cell line A7r5)和活體上(rat gracilis muscle)各設計缺氧-再回氧及缺血-再灌流模型,來研究一氧化氮合成脢在其中的基因表現。初步結果顯示iNOS在缺氧或缺血期可能會造成細胞損傷,在回氧或再灌流期,細胞損傷可能主要由其他的機轉,如游離氧基群或白血球。我們根據有限的數據,提出改善缺血再灌流的假設方法,當然,須要進一步更完整的實驗結果證實。
總之,本論文針對顯微游離組織移植(free tissue transfer)在術前的皮瓣選擇、設計及手術方法,術中、術後血栓及缺血再灌流損傷的三大環環相扣的主題,由基礎的實驗設計(包括in vitro和in vivo)和配合臨床運用,尋求問題的答案,以期病患得到最好的重建結果。

The reconstruction of tissue defects, such as skin, bone, nerve, vessel, muscle, and tendon, is one of the most frequent and devastating problems encountered by a plastic surgeon. The advancement of microsurgical techniques has made simultaneous complex tissue reconstruction possible. Nevertheless, three major difficult issues remain to be solved. The first relates to the choice and design of flap, in order to achieve optimal results with minimal donor-site morbidity. The second is the possibility of vessel thrombosis after microvascular anastomosis, leading to flap failure. The last is the risk of ischemia-reperfusion injury, either locally or systemically, once blood flow is restored after prolonged ischemia. These issues must all be resolved if the operation is to succeed.
The perforator flap has become more widely utilized recently, because it provides for more accurate reconstruction and minimal donor-site morbidity. We carried out cadaver dissection in the laboratory to study in detail the anatomy of the anterolateral thigh flap, one of the perforator flaps. This provided us with the anatomic basis for analyzing the vascular variations in clinical patients, the most troublesome aspect of harvesting this flap. The anatomic variations of cutaneous perforators were investigated and classified into four types. Some technical considerations to facilitate easier flap elevation were also summarized. This flap has been widely applied clinically for reconstruction of head & neck, trunk, and extremities.
Vessel occlusion after microvascular anastomosis results in flap necrosis. Many thrombolytic agents have been applied clinically for salvaging the circulation-compromised free flap. However, the tendency to systemic bleeding and local reocclusion are unsolved problems. It has been shown that the major components of the microvascular thrombus are platelet-rich plugs. Therefore, the ideal thrombolytic agent would be stable, clot-targeted, and without complications of systemic bleeding and local reocclusion. Recombinant DNA technology has been applied in our lab to construct a double mutant streptokinase SK(16-378)-K59AK332A for improvement of stability, and a fusion protein streptokinase SK(16-378)-K59AK332A-γ-(Gly)7-RGDS for better specificity to the platelet-rich clot. The in vitro study showed that SK did indeed have better stability in the former, and better affinity to platelets in the latter. Nevertheless, the in vitro study is not always comparable with that of the complex situation in vivo. A reliable in vivo platelet-rich thrombosis model is mandatory. We attempted to establish a platelet-rich arterial thrombosis model in rabbits and completed some preliminary in vivo tests of recombinant streptokinase proteins.
Once the blood flow is restored after prolonged ischemia, it can induce cellular damage, referred to as "ischemia-reperfusion injury". This results in tissue fibrosis or systemic toxicity. We attempted to design an in vitro experiment with rat VSM cell line A7r5 and an in vivo model with rat gracilis muscle for evaluation of the role of NO (nitric oxide) in hypoxia-reoxygenation and ischemia-reperfusion injury. Expression of the nNOS, eNOS, and iNOS genes was assessed. The preliminary results revealed that NO was deleterious in the hypoxic or ischemic stage. However, in the reoxygenation or reperfusion stage, the cell damage might be dominated by another effector, such as oxygen free radicals or neutrophils. According to the limited data shown in the experiment, we also postulate a defense mechanism against ischemia-reperfusion injury.
In summary, we designed and carried out laboratory cadaver dissection of the cutaneous perforator of the anterolateral thigh flap to enable easier flap harvesting when the dissection was widely applied clinically. The establishment of a platelet-rich thrombosis model provided a reliable in vivo model to study thrombolytic agents, such as recombinant streptokinase proteins. The model is useful for researchers to investigate the biological function of thrombolytic agents in vivo, and also for solving the significant problem of vessel thrombosis after free flap surgery. Moreover, the in vitro and in vivo study of NOS gene expression showed that NO played a negative role in the hypoxic or ischemic stage. We also propose a possible mechanism for ameliorating ischemia-reperfusion injury. Only when the three major hurdles discussed are all overcome, can the patient get the best reconstructive results.

目次 頁數
中文摘要 ---------------------------------------------------------------------- iii
英文摘要 ---------------------------------------------------------------------- v
誌謝 ---------------------------------------------------------------------------- vii
圖目錄 ------------------------------------------------------------------------- ix
表目錄 ------------------------------------------------------------------------- xii
緒論 ----------------------------------------------------------------------------- 1
1-1 顯微重建手術的發展及臨床遭遇的問題 ------------------------- 2
1-2 臨床上理想之重建皮瓣的選擇 ------------------------------------- 4
1-3 溶解富含血小板動脈血栓之活體模型 ---------------------------- 5
1-4 一氧化氮合成脢與缺血再灌流損傷關係的研究 ---------------- 8
穿通枝皮瓣之遺體大體解剖研究及臨床顯微重建的應用 ------ 13
2-1 研究目的 ---------------------------------------------------------------- 14
2-2 研究材料與方法 ------------------------------------------------------- 15
2-3 研究結果 ---------------------------------------------------------------- 20
2-4 討論 ---------------------------------------------------------------------- 23
蛋白in vitro和in vivo之溶血栓測試 --------------------------------- 27
3-1 研究目的 ---------------------------------------------------------------- 28
3-2 研究材料與方法 ------------------------------------------------------- 30
3-3 研究結果 ---------------------------------------------------------------- 54
3-4 討論 ---------------------------------------------------------------------- 58
一氧化氮合成脢在缺氧細胞(in vitro)及缺血再灌流肌肉皮瓣
(in vivo)的基因調控機轉研究 ----------------------------------------- 71
4-1 研究目的 ---------------------------------------------------------------- 72
4-2 研究材料與方法 ------------------------------------------------------- 74
4-3 研究結果 ---------------------------------------------------------------- 87
4-4 討論 ---------------------------------------------------------------------- 91
第五章 結論與展望 -------------------------------------------------------- 97
第六章 參考文獻 ----------------------------------------------------------- 101
第七章 圖與表 -------------------------------------------------------------- 134
第八章 附錄 ----------------------------------------------------------------- 191
自述 --------------------------------------------------------------------------- 192
已發表的相關論文 --------------------------------------------------------- 194

第一章 緒論
1-1 顯微重建手術的發展及臨床遭遇的問題
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1-3 溶解富含血小板動脈血栓之活體模型的研究
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1-4 一氧化氮合成脢在缺氧細胞(in vitro)及缺血再灌流肌肉皮瓣(in vivo)的基因調控機轉研究
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第二章 穿通枝皮瓣之遺體大體解剖研究及臨床重建顯微手術的應用
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第四章 一氧化氮合成脢在缺氧細胞(in vitro)及缺血再灌流肌肉皮瓣(in vivo)的基因調控機轉研究
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第五章 結論與展望
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