臺灣博碩士論文加值系統

Recombinant tissue plasminogen activator (rt-PA)可溶解血栓而使用在急性缺血性腦中風的病患，是近年來在治療腦中風的處置上，一個很重要的發展。然而，rt-PA的治療效益往往因溶解血栓而具有誘發出血的風險。標的運送血栓溶解劑至栓塞之處的應用可以大大降低出血的副作用。因此，我們建立了有利於磁導的大鼠血栓栓塞的模型，作為未來磁導血栓溶解劑之研究。在已麻醉的大鼠中，2 x 1.5 mm的全血血栓由右髂動脈插管處被注射入動脈內，並阻塞在左髂動脈、股動脈及陰腹上動脈的交接處，血流的變化將由超音波及雷射杜卜勒血流計所偵測。經動脈給予rt-PA 及其衍生物human tissue urinary plasminogen activator (HTUPA; 1 mg/kg)後，於30分鐘後即可觀察左髂動脈血流分別回復至近69 %及62 %，且下肢皮膚灌流亦顯著回復。而在左髂動脈上方放置NdFeB 磁鐵 15 分鐘，經Prussian blue組織染色結果發現，nanomag®-D (250 nm; 0.1 and 1 mg/kg)在動脈裡無論有無血栓存在，皆可對抗血液動力學的拉力而滯留。分別經動、靜脈給予nanomag®-D、PPy、d-PPy、PAn、d-PAn USPIO-102 (1 mg/kg)並不會導致局部血液動力學的變化及angiotensin II 和 acetylcholine對於血管的反應 。另外，於大鼠提睪肌微循環中於磁場存在下給予nanomag®-D不影響微循環血流，而經腎動脈給予nanomag®-D 、PPy、d-PPy、PAn及d-PAn在無磁場存在下皆不會聚集沉積在腎臟組織。然而 ，以nanomag®-D (30-100 μg/ml) 、PAn及d-PAn (100-300 μg/ml)處理血管平滑肌細胞的2-6天中，可抑制細胞增生，而PPy、d-PPy則不影響細胞增生。總結上述結果我們的實驗證明大鼠血栓栓塞模型能被應用於急性標的磁導血栓溶解，然而這些磁性奈米粒子在長期大量應用時所造成的細胞毒性，亦是不容忽視的問題。

The use of recombinant tissue plasminogen activator (rt-PA) to cause thrombolysis has been an important development in the treatment of ischemic stroke. However, rt-PA exhibits potential to induce hemorrhage as its major side effect. Targeted delivery of thrombolytic agents to the site of thrombus may greatly reduce the side effect and amount of the drug used. In the current study, we developed a rat embolic model for evaluation of magnetic targeted delivery of thrombolytic agents. In anesthetized rats, a whole blood clot was injected from a retrograde catheter in the right iliac artery and lodged in the left iliac artery before branching into femoral and pudic epigastric arteries. The hemodynamic changes in response to clot lodging and thrombolysis were monitored with ultrasonic and laser Doppler flowmetry. Thirty min after intra-arterial infusion of recombinant tissue plasminogen activator (rt-PA) and human tissue urinary plasminogen activator (HTUPA) reversed approximately 69 and 62 % of the iliac blood flow and most of the skin perfusion of the left hind limb. Placement of an NdFeB magnet above the left iliac artery for 15 min caused intra-arterial retention of magnetic nanoparticle nanomag®-D (250 nm; 0.1 and 1 mg/kg) against hemodynamic dragging force in the presence and absence of the clot, as illustrated by histological staining with Prussian blue. Intra-arterial or intravenous injection of nanomag®-D, PPy, d-PPy, PAn, d-PAn or USPIO-102 (1 mg/kg) did not alter basal hemodynamics or hemodynamic responses to angiotensin II or acetylcholine. In addition, injection of nanomag®-D to the microcirculation of cremaster muscle of rat did not alter the blood flow. Intra-arterial injection of nanomag®-D, PPy, d-PPy, PAn or d-PAn did not cause retention in the renal tissues without magnetic field. Nevertheless, incubation of nanomag®-D (30-100 μg/ml), PAn and d-PAn (100-300 μg/ml) with cultured vascular smooth muscle cells for 2-6 days did suppress cell proliferation. In conclusion, we have demonstrated a rat embolic model that can be used for acute magnetic targeted delivery of thrmobolytic drugs; however, potential long term toxicity of these nanoparticles can not be ignored.

目錄 (Contents)
指導教授同意書……………………………………………………….
口試委員會審定書…………………………………………………….
授權書………………………………………………………………….iii.
誌謝…………………………………………………………………….iv
中文摘要……………………………………………………………….v
英文摘要………………………………………………………………vii
目錄…………………………………………………………………….xi
縮寫表………………………………………………………………….xii
第一章 緒論(Introduction)
1.1：背景及相關研究…………………………………………………1
1.1.1：t-PA之結構…………………………………………………….1
1.1.2：t-PA在急性缺血性腦中風的角色…………………………….2
1.1.3：與t-PA相關的其他凝血因子……………………..…………..5
1.1.3.1：t-PA與凝血酶活化纖維溶解抑制劑(TAFI, thrombin –
activatable fibrinolysis inhibitor)………………………..6
1.1.3.2：t-PA與胞漿素原活化酶抑制劑-1………………….......7
1.1.3.3：t-PA與蛋白C……………………………………….......7
1.1.4：磁性奈米粒子 (MNP, Magnetic nanoparticle)………………...9
1.1.4.1： 細胞磁性分離…………………………………………11
1.1.4.2：惡性腫瘤細胞的熱治療………………………………..11
1.1.4.3：核磁共振影像…………………………………………..12
1.1.4.4：藥物運送………………………………………………..12
1.1.5磁性標的載體(Magnetic Targeted Carrier, MTC)….….……….14
1.2 實驗目的…………………………………………………………20
第二章 實驗材料與方法(Materials and Methods)
2.1：實驗材料………………………………………………………..21
2.2：實驗方法………………………………………………………..22
2.2.1: 大鼠的預備與手術……………………………………..23
2.2.2: 腎動脈給藥……………………………………………..28
2.2.3: 組織切片與染色………………………………………..29
2.2.4: 血栓完整性評估………………………………………..29
2.2.5: 雷射杜卜勒血流影像分析……………………………..30
2.2.6: 在微循環的動物模型中觀察磁性奈米粒子變化分佈..30
2.2.7: 細胞製備………………………………………………..31
2.2.8: 細胞計數………………………………………………..32
2.2.9: PPy及PAn的合成……………………………………...35
2.2.10: 分析及統計方法………………………………………37
第三章：實驗結果(Results)……………….…………………………38
第四章：討論(Discussion)………………………………………..49
第五章：結論(Conclusion)……………………………………….57
未來展望……………………………………………………………...58
圖(Figure)…………………………………………………………60
參考文獻……………………………………………………………...75

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