http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/146 2026-03-14T13:53:49Z 2026-03-14T13:53:49Z Ying-Ru Chen 陳盈如 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64713 2021-06-16T22:57:55Z 2012-01-01T00:00:00Z

標題: 龜殼花蛇毒蛋白Disintegrin抗血栓活性與作用機轉之探討; The Antithrombotic Effects and Mechanisms of Disintegrin Purified from Trimeresurus mucrosquamatus Snake Venom 作者: Ying-Ru Chen; 陳盈如 摘要: Disintegrins是小分子量的抗血栓蛋白，最早由蛇毒蛋白中所發現，並有著強效的血小板凝集抑制活性。Disintegrins的作用機轉是抑制血小板上的GPIIb/IIIa，被認為具有開發為抗血栓抑制劑的潛力。龜殼花 (Trimeresurus mucrosquamatus) 蛇毒蛋白原毒粉末依序經由CM-Sephadex C-50離子交換層析法、Sephadex G-75 膠質過濾法、FPLC Superdex-75膠質過濾法，最後再以逆向高效液相色譜分析法進行最後純化分離，純化得到的兩種disintegrin分別為TMV-2和TMV-7。在本篇研究中，主要的目的是探討並比較TMV-2和TMV-7抗血栓作用和機轉不同之處。TMV-2和TMV-7有著不一樣的等電點，TMV-2的等電點約為4.5而TMV-7的等電點約為5-7。TMV-2和TMV-7都是單鏈的小分子，分子量經由MALDI-TOF測定分別為7663 和7672 Da。由LC-MS/MS所鑑定出的部分序列顯示，TMV-2有24%的序列相似於batroxostatin (由Bothrops atrox蛇毒蛋白中分離出的disintegrin)；而TMV-7也有24%的序列相似於batroxostatin，但有高達79%的序列相似於cotiarin (由Bothrops cotiara蛇毒蛋白中分離出的disintegrin)。TMV-2和TMV-7都能有效抑制在人類富含血小板的血漿、人類血小板懸浮液以及經elastase處理的人類血小板懸浮液中所引發的凝集反應，其抑制的程度會隨著濃度上升而增大。TMV-2和TMV-7都不會影響thromboxane A2的生成，但都會增強由collagen和thrombin所引起的P-selectin的表現。利用流式細胞儀的分析，可觀察到TMV-2對於GPIIb/IIIa的單株抗體7E3結合到GPIIb/IIIa上有明顯抑制作用，然而TMV-7卻不會抑制單株抗體7E3結合到GPIIb/IIIa上。而TMV-2和TMV-7對於另一種GPIIb/IIIa的單株抗體10E5的影響卻是會加強此單株抗體結合到GPIIb/IIIa上。在動物實驗方面，TMV-2和TMV-7也能抑制老鼠富含血小板的血漿所中所引起的血小板凝集反應，其抑制的程度也能隨著濃度上升而增大。在給予老鼠0.25μg/g的劑量之下，TMV-2會顯著的延長出血時間，然而TMV-7則不會延長出血時間。另外，TMV-2和TMV-7兩者都不會減少血小板的數量。綜合上述結果可知，TMV-2和TMV-7這兩個等電點不相同的disintegrin有著不一樣的特性。TMV-2比起TMV-7有著更強效的血小板凝集抑制效果，而且TMV-7結合到GPIIb/IIIa上的位置顯然和TMV-2及單株抗體7E3不同。另外，我們發現當TMV-2或TMV-7和單株抗體10E5一起作用時反而會引發血小板的活化。而給予老鼠0.25μg/g的劑量之下，TMV-7的副作用和TMV-2比起來顯得較少。由此我們認為TMV-2和TMV-7這兩種disintegrin之間的差異研究可利於新一代GPIIb/IIIa拮抗劑的研發，而且由TMV-2和TMV-7作用於GPIIb/IIIa的不同之處或許可以提供訊息來探討GPIIb/IIIa拮抗劑所引發血小板減少症的機轉。; Disintegrins are small moleculars and potent platelet inhibitors found in the snake venom. Disintegrins are GPIIb/IIIa antagonists and potential antithrombotic agents. By means of CM-Sephadex C-50, Sephadex G-75 gel filtration, FPLC Superdex 75 gel filtration and reverse phase HPLC, two disintegrins, TMV-2 and TMV-7, were purified from Trimeresurus mucrosquamatus snake venom. In this study, we investigated and compared the difference between TMV-2 and TMV-7. TMV-2 and TMV-7 had different isoelectric point (pI). The pI of TMV-2 was estimated to be around 4.5, whereas that of TMV-7 was estimated to be around 5-7. TMV-2 and TMV-7 were shown to be a single peptide chain. By MALDI-TOF, the molecular weigh of TMV-2 and TMV-7 were determined as 7663 and 7672 Da, respectively. The sequence of TMV-2 was 24% identical to batroxostatin, a disintegrin purified from the snake venom of Bothrops atrox, whereas the sequence of TMV-7 was 24% identical to batroxostatin and 79% identical to cotiarin, a disintegrin purified from the snake venom of Bothrops cotiara. Both TMV-2 and TMV-7 concentration-dependently inhibited platelet aggregation in human platelet-rich plasma, washed human platelet suspension and elastase-treated human platelets. Also, TMV-2 and TMV-7 did not interfere with the formation of thromboxane A2. However, both TMV-2 and TMV-7 enhanced the P-selectin expression induced by collagen and thrombin. In the indirect binding assay, TMV-2 significantly inhibited 7E3, a mAb raised against GPIIb/IIIa, binding to GPIIb/IIIa, but TMV-7 did not. Both TMV-2 and TMV-7 enhanced 10E5, a mAb raised against GPIIb/IIIa, binding to GPIIb/IIIa. In the animal models, TMV-2 and TMV-7 dose-dependently inhibited platelet aggregation in mice PRP. Furthemore, TMV-2 (0.25μg/g) prolonged the bleeding time more significantly than TMV-7 (0.25μg/g) as they were intravenously administered. However, both TMV-2 and TMV-7 did not alter the platelet counts. In conclusion, TMV-2 and TMV-7, two disintegrins with different isoelectric point, have the different characters. Although both TMV-2 and TMV-7 are GPIIb/IIIa inhibitor, the inhibitory effect of TMV-2 was more potent than TMV-7. Also, our data revealed that the binding site of TMV-7 on GPIIb/IIIa was different from TMV-2 and mAb 7E3. Moreover, combination of TMV-2 or TMV-7 with mAb 10E5 could lead to platelet activation. Finally, we found that TMV-7 had fewer side effects than TMV-2 at the same dosage. Therefore, the difference between TMV-2 and TMV-7 may provide valuable information for the development of GPIIb/IIIa inhibitors and the mechanisms involved in GPIIb/IIIa inhibitor-induced thrombocytopenia.

2012-01-01T00:00:00Z Chih-Peng Lin 林至芃 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/3891 2021-05-13T08:38:06Z 2016-01-01T00:00:00Z

標題: 鴉片類止痛藥物耐受性的轉譯研究: 從臨床到基礎; Translational Research on Opioid Analgesic Tolerance: from Bedside back to Bench 作者: Chih-Peng Lin; 林至芃 摘要: 鴉片類藥物是臨床處理中重度疼痛，最有效也最被廣泛使用的藥物。然而長期使用鴉片類藥物止痛卻也會伴隨產生藥物的耐受性。脊椎管內鴉片類藥物投與將藥物直接送至中樞神經產生藥效，是非常有效的止痛方式但卻也更容易產生藥物耐受性。傳統神經科學以神經元為中心的思維下，鴉片類藥物耐受性的致病機轉著重於Opioid receptor的internalization、NMDA receptor的upregulation或glutamate transporter 的down regulation。然而這些現象都只能部分解釋耐受性的成因。最近在齧齒類實驗動物上的研究結果顯示神經膠細胞的活化及發炎性物質的過度表現、也就是所謂的神經炎性反應在神經病變疼痛與類鴉片耐受性的致病機轉上有重要的角色。然而相關的人體研究證據則相當稀少。 在本研究的臨床部分，我們首先嘗試建立國內最完整的，有關體外可程控式植入型脊椎內給藥系統的照護流程。包括合適病患的選取、脊椎內嗎啡藥物輸注測試、手術方式的改進、長期追蹤與品質提升計畫。我們紀錄並分析了流程建立初期的病患，其脊椎管內嗎啡的劑量的改變、治療相關併發症的發生率與後續處理及病患日常生活功能的改善程度。藉由給予足夠劑量的脊椎管內嗎啡，病患的疼痛控制與生活品質皆能大幅改善。然而在這當中我們也發現所有接受脊椎內嗎啡輸注療法的病患，其嗎啡的劑量皆快速的增加，遠遠超過病情的演進。 以臨床的照護觀察到的現象為起點，在研究倫理委員會核准後，我們進行了一系列的轉譯醫學研究。我們首先分析了已產生類鴉片藥物耐受性病患的腦脊髓液中發炎相關因子的濃度。包括TNF-alpha、 CXCL1、CXCL10、CCL2、CX3CL1及CXCL12並與未暴露類鴉片藥物的對照組受試者比較。研究結果發現，已經對類鴉片止痛藥產生耐受性的病患群，其腦脊髓液中的CXCL1及CXCL12濃度明顯高於未暴露類鴉片藥物的對照組。進一步我們更發現CXCL1的濃度與病患所接受的類鴉片止痛藥物劑量成高度正相關。 接著我們建立轉譯動物實驗模式，藉由實驗鼠的閃尾反應，評估嗎啡的止痛效果及相關發炎因子對類鴉片耐受性產生的影響。在實驗大鼠投予嗎啡誘發藥物耐受性後，大鼠脊髓組織之CXCL1 及CXCL12 mRNA皆顯著增加。雖然單獨給予椎管內CXCL1或CXCL12並不會改變老鼠的基礎閃尾行為，然而椎管內給予CXCL1或CXCL12卻會大幅降低腹腔內給予嗎啡所造成的急性止痛效果。接著我們參照臨床長期椎管內類鴉片輸注用於頑固疼痛的處置，建立植入皮下微幫浦進行長期椎管內嗎啡輸注並誘發大鼠產生藥物耐受性的實驗模式。我們發現耐受性的發生會因同時給予嗎啡與CXCL1或CXCL12 而加速。反之 若被嗎啡輸注時一併給予CXCL1或CXCL12的中和抗體則會延緩耐受性的發生。針對CXCL1 訊息傳遞給予其受體CXCR2的拮抗劑antileukinate hexapeptide，或針對CXCL12 訊息傳遞給予其受體CXCR4的拮抗劑 AMD3100，則可更有效的延緩嗎啡耐受性的發生。 綜合以上的實驗結果，我們藉由臨床到實驗動物的轉譯醫學研究模式驗證了趨化激素CXCL1與CXCL12可能參與鴉片類止痛藥耐受性的形成。阻斷 CXCL1/CXCR2 與 CXCL12/CXCR4 的訊息傳遞路徑則可以延緩藥物耐受性的產生並降低其嚴重度。因此針對CXCL1/CXCR2 與 CXCL12/CXCR4 的訊息傳遞路徑進行介入將是治療類鴉片止痛藥耐受性的新藥研發之潛力標的。; Opioid analgesics remain the most effective and widely used analgesics for the management of moderate to severe pain. However, the efficacy of long-term opioid analgesics is progressively attenuated by tolerance, preventing adequate pain relief under stable opioid dosages for chronic pain patients. Although intrathecal opioid delivery provides very effective analgesia by acting directly on central nervous system, opioid analgesic tolerance is also accelerated. Classical neuron-centered concepts such as internalization of opioid receptors, up-regulation of N-methyl-D-aspartate receptor function, or down-regulation of glutamate transporter activity can only partially explain the phenomenon of tolerance. Recent evidence showing glial activation and upregulated inflammatory mediators in the rodent central nervous system has confirmed the pivotal role of neuroinflammation in neuropathic pain or opioid tolerance, or both. However, human evidence is still sparse. In clinical part of this study, we developed comprehensive management protocol for totally implantable programmable intrathecal drug delivery system from patient selection, intraspinal morphine trial, surgical procedure to follow up program. Intrathecal morphine dosage adjustment, treatment related complications and patient functional outcomes are recorded regularly and analyzed. By delivering liberal dose of intrathecal morphine, pain severity decreased significantly. Due to much better pain control and improved quality of life, functional performance status also improved. Intrathecal morphine delivery by using totally implantable programmable device is an effective alternative method to relieve refractory cancer pain. Based on our clinical practice, we further conducted subsequent translational research by investigating the intraspinal cytokine and chemokine profiles of opioid-tolerant cancer patients after research ethic committee approval. Cerebrospinal fluid (CSF) samples from opioid-tolerant cancer patients and opioid-naive subjects were compared. The CSF levels of tumor necrosis factor-alpha, CXCL1, CXCL10, CCL2, CX3CL1 and CXCL12 were assayed. CXCL1 and CXCL12 levels in CSF were significantly upregulated in the opioid-tolerant group. Further analysis revealed that CXCL1 level was strongly positively correlated with opioid dosage. The rat tail flick test was utilized to assess the effects of intrathecal CXCL1 or CXCL12 on morphine-induced acute antinociception and analgesic tolerance. After induction of tolerance by intrathecal morphine infusion, the spinal cord CXCL1 and CXCL12 messenger RNA were significantly upregulated. Although CXCL1 or CXCL12 infusion alone did not affect baseline tail flick latency, the analgesic tolerance was accelerated by intrathecal infusion of CXCL1 or CXCL12 in daily intraperitoneal morphine injection of paradigm. After establishing tolerance by intrathecal continuous infusion of morphine, its development was accelerated by co-administration of CXCL1 or CXCL12. On the contrary, tolerance was attenuated by co-administration of CXCL1 or CXCL12 neutralizing antibody or corresponding receptor antagonists. CXCL1 and CXCL12 were upregulated in both opioid-tolerant patients and rodents. The onset and extent of opioid tolerance was affected by antagonizing intrathecal CXCL1/CXCR2 and CXCL12/CXCR4 signaling. Therefore, the CXCL1/CXCR2 and CXCL12/CXCR4 signal pathways may be novel drug targets for the treatment of opioid tolerance.

2016-01-01T00:00:00Z 葉禹萱 Yu-Hsuan Yeh http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77516 2024-02-27T19:24:56Z 2018-01-01T00:00:00Z

標題: 高表現FOXC1在非小細胞肺癌對EGFR—TKI抗藥性之研究; Role of High FOXC1 Expression in EGFR-TKI Resistant Non-Small Cell Lung Cancer 作者: 葉禹萱; Yu-Hsuan Yeh 摘要: 以EGFR-TKI治療EGFR activating mutation之非小細胞肺癌病人，具相當好的療效，但數個月後病人會產生acquired resistance。轉錄因子FOXC1在癌症中扮演重要角色，我們發現，在EGFR-TKI抗藥性細胞中，其表現增加。本篇論文使用H1975與H1975/AR (AZD9291 resistance) 細胞探討FOXC1在EGFR-TKI抗藥性之角色。 過量表現FOXC1於H1975細胞，可增進細胞之生長速度、遷移能力與形成球體（sphere）癌幹細胞，FOXC1亦調控癌幹細胞基因ALDH1A1之表現。在H1975/AR細胞抑制FOXC1之表現會促進RhoGDI2轉錄；在H1975細胞抑制RhoGDI2，會促進細胞之遷移能力與癌幹細胞特性。因此ALDH1A1與RhoGDI2可能均為FOXC1之下游標的。在原位肺癌動物模式發現，高表現FOXC1可促進腫瘤生長；以Kaplan Meier Plotter分析，FOXC1高表現之肺腺癌病人整體存活率較差。這些發現推測FOXC1可能與EGFR-TKI之抗藥性有關，可成為治療非小細胞肺癌的一個預後標記。; EGFR tyrosine kinase inhibitors (EGFR-TKIs) have shown good efficacy in non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, acquired resistance develops after several months of treatment. Forkhead Box C1 (FOXC1) is an important transcriptional regulator associated with a wide variety of carcinomas including NSCLC. In this study, we found the overexpression of FOXC1 in EGFR-TKI resistant cells. Its role in the resistance of EGFR-TKIs was investigated using H1975 and AZD9291-resistant H1975/AR cell lines. Overexpression of FOXC1 in H1975 cells led to an increase in cell proliferation, migration and sphere-forming ability. In addition, FOXC1-overexpressing cells showed an up-regulation of ALDH1A1. On the other hand, knockdown of FOXC1 in H1975/AR cells showed an up-regulation of RhoGDI2. Further analysis demonstrated that knockdown of RhoGDI2 in H1975 cells increased cell migration and sphere-forming ability. Therefore ALDH1A1 and RhoGDI2 might be the downstream targets of FOXC1. Furthermore, overexpression of FOXC1 increased tumor growth in orthotopic lung cancer model. In Kaplan Meier Plotter database, high expression of FOXC1 is related to poor overall survival in lung adenocarcinoma. These findings indicate that FOXC1 might involve in the resistance of EGFR-TKIs and provide a possible prognostic marker for the treatment of NSCLC.

2018-01-01T00:00:00Z Tsai-Yueh Luo 羅彩月 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39453 2021-06-13T17:28:53Z 2004-01-01T00:00:00Z

標題: 高血脂兔心肌功能之研究及肝癌治療用核醫藥物之開發; A Study of Cardiac Function in the Hypercholesterolemia Rabbits and the Development of Therapeutic Radiopharmaceuticals for Hepatic Tumors 作者: Tsai-Yueh Luo; 羅彩月 摘要: 國人飲食及生活習慣的改變，使心血管疾病亦隨著歐美國家之腳步位居國人十大死因之前三名，其中以動脈粥狀硬化則為引起心血管疾病及死亡之主因，而高膽固醇血症則是血管粥狀硬化的重要元兇之一。有鑑於此，對高膽固醇的致病生理機轉做深入之瞭解實有其必要性。 由於高膽固醇與動脈粥狀硬化之關係已有許多詳細之研究，但有關高膽固醇對心肌交感神經之影響則甚少報告。本研究第一部份之研究重點在於: 探討高膽固醇食物對心臟交感神經功能之影響。我們選用三個月大之紐西蘭白兔做為實驗動物，將其分成二組，一組餵食普通之飼料(稱為控制組)，另一組則給予高膽固醇食物(稱為高膽固醇組)，在餵食前與餵食三個月後，分別注射123I-MIBG (123I-meta-iodobenzylguanidine)後，以核子醫學方法評估活體動物之交感神經功能。MIBG與臨床常用藥物Guanethidine之化學結構類似，已被證實與交感神經分泌物Norepinephrine(NE)利用相同神經再回收路徑及貯存之方法，當MIBG標幟上123I後，可用於診斷許多器官(包括心臟等)之交感神經完整性(integrity)及功能。經過三個月之餵食後，將所有的兔子犧牲，取其右心室乳突肌，分別以Isoproterenol(ISO)及NE評估其對心肌收縮力之增強作用。 實驗結果發現，高膽固醇組較同年齡之控制組有較高之心肌MIBG回收率，呈現較高之心肌對縱膈腔回收比值顯示其有較高之交感神經密度。123I-MIBG之心肌清除比值反應其交感神經之活性，高膽固醇組僅呈現較控制組稍微高但不具統計意義。心室肌肉等長收縮反應之試驗方面，發現ISO之濃度-反應曲線圖比起控制組明顯向右飄移。另外，我們將部份心室乳突肌先以cocaine前處理30分鐘使其阻斷交感神經未稍之回收功能後，與未經cocaine處理之乳突肌比較兩者對NE收縮反應之差異，發現經cocaine處理後之心肌，高膽固醇組之NE反應明顯較控制組大。針對本試驗之結果，我們的結論是兔子之高膽固醇血症會導致心肌之交感神經密度增加，且降低ISO的增強收縮力之作用，但在cocaine處理之心室乳突肌則會更增強其對NE之反應，本實驗結果顯示高膽固醇血症會引起神經重建(neuronal remodeling)，改變心臟對交感神經受體致活性之反應。 高膽固醇血症對心臟功能之影響除了導致血管粥狀動脈硬化外，並會導致心肌細胞膜之膽固醇含量增加而造成細胞膜結構或功能之改變。因此，本論文中除了探討交感神經系統功能之變化外，第二部份重點在於:探討不同胞外鈣離子濃度及不同刺激頻率時右心室乳突肌收縮力之表現，同時分析右心室細胞之鈣管道表現，並研究高膽固醇血症對細胞內之鈣離子調控基因及其相對蛋白的表現之影響。我們發現在胞外鈣離子濃度為2 mM並給予3 Hz之刺激頻率時，高膽固醇組之心室乳突肌收縮力較控制組弱，同時達到最大收縮力所需時間亦較控制組長。分析兩組之最大L-型鈣電流密度，高膽固醇組僅稍高於控制組但不具統計意義。以RT-PCR分析其mRNA的表現量，並以GAPDH的mRNA值將之標準化後，發現高膽固醇組sarcoplasmic reticulum Ca2+- ATPase (SERCA)的表現明顯低於控制組。同樣地，ryanodine receptor (RyR)亦有類似之結果。但相反地，NCX則在高膽固醇組表現量較高。Western blotting之實驗結果顯示SERCA的蛋白表現量降低而Na+-Ca2+ exchanger (NCX)則增加，另外dihydropyridine receptor的表現兩組間並無差異。 由上述心肌功能之研究發現，高膽固醇血症不只降低心肌等長收縮力之表現，同時也延長其收縮達到最大峰值之時間。在心室細胞之膜電位鉗定試驗中，我們也發現高膽固醇組之鈣電流密度僅稍大於控制組(但不具統計意義)且其鈣管道不活化曲線並不受高膽固醇之影響。由於高膽固醇對鈣電流之影響極微，因此，我們認為(1)SERCA及RyR之減少(2)NCX之增加，與高膽固醇血症會抑制最大心肌收縮力及延緩收縮力到達峰值之時間有密切關係。 另外，根據行政院衛生署的統計資料，除了心血管疾病外，癌症更是國人十大死因之首，開發肝癌之治療用藥更有其急迫性。對肝癌之治療雖然外科手術一般被認為是治療之首選方式，但一般而言，肝細胞癌之預後皆不樂觀，其它之治療方法例如化學療法、放射治療及經肝動脈栓塞結果也都不令人滿意。另一治療之選擇即為利用Lipiodol當作載體(carrier)可以攜帶化學治療用藥物或放射性同位素當作栓塞治療之用途則為目前國內外研究的焦點。因此，本研究之第三部份重點在於: 肝癌治療用藥物之開發研究。 Lipiodol是由罌粟子油提煉出來的一種碘化脂肪酸，Nakamura等人的實驗發現Lipiodol滯留於肝腫瘤相當長的時間，可用於肝腫瘤的診斷或栓塞治療。188Re為新近研發成功的治療用核種，它的半衰期(half-life)短，及放射能量適中，非常適合於放射治療與療程追蹤。99mTc-ECD(ethyl cyteinate dimer)為一臨床已使用的腦血流灌注造影劑，我們利用Tc化性與Re近似，且其配位子ECD具有親脂之特性，嘗試進行188Re標幟ECD之試驗以並發展Lipiodol之劑型，形成188Re- ECD/Lipiodol混合物，並植入肝癌細胞之大白鼠評估其是否具有肝癌治療之潛力。 依據我們目前之處方研究，比較好之188Re-ECD標幟方法為:(1)tartaric acid (TA)較EDTA更適於當螯合劑(chelating agent)；(2)每一反應瓶內含1 mg ECD, 15 mg SnCl2及5-10 mg TA。在上述條件下，可得約80%之標幟效率，而後經萃取及離心等步驟處理後，終產品之放射化學純度可超過94%。由生體分佈資料顯示肝動脈注射方式給予之188Re-ECD/Lipiodol主要分佈於肝腫瘤區域及正常之肝組織，兩者之Target/Nontarget比值由第一小時之13.21而後緩慢降低至第四十八小時約為6.84。在本研究中，我們發現利用體外系統測試188Re-ECD發現其可穩存於親脂性溶液之Lipiodol內。但是在體內時，仍有部份之188Re-ECD被游離出來，可能受到血液中特別之酯類分解酵素(esterase)代謝使其由血液及非腦部區域清除。由動物實驗生體分佈數據得知，肝動脈給藥之方式可以使188Re-ECD/Lipiodol在肝腫瘤區域有較高之放射性分佈，而在其它器官則相對僅有少量活性分佈。我們認為188Re-ECD/Lipiodol具有製備簡單之優點，同時具放射活性可較長時間停留腫瘤組織，未來具潛力發展成為肝腫瘤治療藥物之一。; Hypercholesterolemia is a risk factor commonly associated with atherosclerosis, coronary artery disease, and myocardial infarction. In order to assess the effect of hypercholesterolemia on cardiac sympathetic nerve function, New Zealand white rabbits were fed a normal diet (control group), or one enriched with 0.5% cholesterol (hypercholesterol group, HC group) for 3 months. Before and after the 3-month diet treatment, we performed serial imaging examinations and analyzed the uptake and washout ratio of 123I-meta-iodobenzylguanidine (123I-MIBG) from the myocardium by administration of 123I-MIBG through an ear vein. MIBG is an analog of guanethidine that shares the same neuronal uptake transport and storage mechanisms as norepinephrine (NE). When labeled with iodine-123, it can be used to image the integrity and function of adrenergic neurons in many organs, including the heart, with conventional planar or single photon emission computed tomography techniques. At the end of the experiments, the rabbits were sacrificed, and right ventricular strips were taken from their hearts. The inotropic response of the right ventricular strips to isoproterenol (ISO) and NE were evaluated. The cardiac MIBG uptake of the HC group, which is evaluated by using the heart/mediastinum (H/M) ratio, was higher than that of the age-matched control group. However, the washout ratios of 123I-MIBG did not statistically differ between the 2 groups. On pretreatment with cocaine, NE-enhanced contractility was greater in papillary muscles isolated from the HC group. The concentration-response curve to ISO was shifted to the right in the HC group, compared with that in the control group. In conclusion, hypercholesterolemia in rabbits resulted in an increase in sympathetic nerve density in the myocardium, a decrease in the inotropic response to ISO, and an increase in the inotropic response to NE in cocaine-treated myocardium. Both the in vivo and in vitro studies demonstrated the functional significance of neural remodeling induced by hypercholesterolemia. Although hypercholesterolemia resulted in a change of sympathetic function, little is known about the effect of hypercholesterolemia on cardiac contractile function. The objective of the further study was to examine the effect of hypercholesterolemia on myocardial contractility. Fifteen New Zealand white rabbits were fed with standard chow (control group) and the other 15 with cholesterol-enriched diet (HC group) for 12 weeks. The contractile response of ventricular muscle strips was measured in various extracellular calcium concentrations and at different pacing rate. The whole cell calcium current recording, mRNA and protein levels of cellular calcium handling proteins were also analyzed. In 2 mM Ca2+ and stimulated at 3 Hz, the contractile force of HC strips was less than that of the controls. The time to peak tension was longer in HC strips. Peak L-type calcium inward current density was slightly higher but not statistically significant in HC myocytes. The mRNA level of sarcoplasmic reticulum Ca+2-ATPase (SERCA) was significantly lower in the HC than that in controls, so was the ryanodine receptor (RyR). The mRNA of Na+/Ca+2 exchanger (NCX) was statistically higher in the HC group. Western blot experiments revealed that protein expression of SERCA in the HC strips decreased, but that of NCX increased. The protein expression of dihydropyridine receptor (DHPR) was similar between these two groups. From the myocardial functional experiment, it was shown that hypercholesterolemia not only decreased the isometric contractile force but also prolonged its time to peak tension. In the voltage-clamp experiments of ventricular myocytes, hyperchol- esterol emia slightly enhanced the L-type calcium current but had no effect on the inactivation curve. Because of little effect on calcium current, we conclude that the suppression of maximal contractile function and the prolongation of systolic contractile time course in hypercholesterolemia are probably mediated by a decreased SERCA and RyR and an increased NCX expression. In the third part of this dissertation, we tried to design a new hepatoma treatment agent by using a therapeutic isotope to label lipophilic ligand-ECD (ethyl cyteinate dimer). Primary hepato- cellular carcinoma (HCC) is the most common form of primary hepatic carcinoma, particularly in Asia and sub-Sahara Africa. Although surgery is usually considered the treatment of choice, the prognosis of HCC is very low. Other therapeutic modalities such as chemotherapy, radiotherapy and trans-hepatic arterial embolization (TAE) showed unsatisfactory results. Alternative methods of therapy using Lipiodol as a carrier for chemotherapeutic agents or radioisotopes are currently under investigation. Radiolabeled Lipiodol has been used in hepatoma therapy. In this report, we attempted to develop a new 188Re-ECD/Lipiodol radiopharmaceutical in which the chelating agent, ECD, is the constituent of the known brain perfusion agent, 99mTc-ECD, and to evaluate its stability and biodistribution in rats with hepatic tumors. Firstly, 188Re-ECD was prepared in a vial, followed by extraction with Lipiodol to get the final product, 188Re-ECD/Lipiodol. The optimal labeling conditions for 188Re-ECD were: (1) tartaric acid which is better than EDTA as a weak chelating agent; and (2) 15 mg of SnCl2, as the reducing agent, and 5-10 mg of tartaric acid in each vial had a better labeling yield. The radiochemical purity of 188Re-ECD/Lipiodol was more than 94 %. Twenty-four male Sprague-Dawley rats with liver tumors were sacrificed at 1, 24, and 48 hrs (eight rats each time) after an injection of approximately 7.4 MBq of 188Re-ECD/Lipiodol via the hepatic artery. The radioactivity in the liver tumor is significant higher following therapeutic arterial injection, and relatively low in other organs including the bone, spleen, brain, thyroid, stomach, muscle, blood and testis throughout this study. In conclusion, the new preparation of 188Re-ECD/Lipiodol is a candidate agent for the treatment of liver cancer.

2004-01-01T00:00:00Z