http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/118 Tue, 24 Mar 2026 19:53:14 GMT 2026-03-24T19:53:14Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31938 標題: Ｔ細胞第四因子(Tcf4)與細胞死亡相關蛋白(Daxx)結合作用之探討; Studies on the interaction between T cell factor 4 (Tcf4) and cell death-associated protein (Daxx) 作者: Shu-Ling Tzeng; 曾淑玲 摘要: 近年來的研究報導指出，在人類大腸癌細胞株細胞中，因為抑癌基因APC的突變或是乙型卡特寧本身的突變，導致細胞核內的人類T細胞因子與乙型卡特寧蛋白複合物活性的持續活化，因而造成人類T細胞因子所調節的下游基因失控是造成大腸上皮細胞的轉型以及息肉生成的主要原因。事實上，人類T細胞因子與乙型卡特寧蛋白複合物對轉譯活性的調節，不管是在胚胎發育或是癌症生成的過程中都存在著相當的意義。在嘗試探討T細胞因子在癌症生成過程中所可能扮演的角色之前，必須先釐清T細胞因子的調控網絡如何運作。雖然T細胞因子可以透過HMG直接與去氧核醣核酸結合，然而這樣的結合在缺乏與乙型卡特寧結合形成蛋白複合體時，T細胞因子並不足以獨立啟動其下游基因的轉譯，除此之外，更多的實驗結果顯示乙型卡特寧不只有如上所述與T細胞因子形成蛋白質複合體以啟動下游基因的轉譯活性，同時也可以將T細胞因子對下游基因的調控功能，由原本的轉譯抑制轉換為轉譯活化，鑑於對相同下游基因的不同作用，科學家們深信必定存在有一嚴謹的開關，得以嚴密調控並且決定細胞發育生長過程中不同的命運。 在T細胞因子轉譯活性的調控中，可能存在與T細胞因子結合的介質蛋白以及其彼此之間交互作用的機轉激發我們相當的研究興趣。由於在人類大腸上皮細胞中存在大量的T細胞第四因子蛋白，因此實驗設計以T細胞第四因子為餌，透過酵母菌雜交的方式順利分離出可能與T細胞第四因子結合的候選蛋白-人類細胞死亡相關蛋白：Daxx。 Daxx蛋白最早被發現是在細胞質中扮演Fas/JNK相關訊息傳遞的媒介蛋白，不過近年來更多的證據指出，其實Daxx蛋白主要是存在細胞核中，扮演基因轉譯活性的調節者。藉由在人類293T細胞中的共同免疫沈澱法以及在酵母菌Y190菌種中的雜交法，證實了Daxx蛋白與T細胞第四因子蛋白之間的結合作用，並且定出T細胞第四因子與Daxx蛋白的結合區間。在細胞核內，Daxx蛋白藉由減低T細胞第四因子與去氧核醣核酸結合的能力而達到抑制T細胞第四因子轉譯活性。因此，在人類大腸癌細胞中大量表現Daxx蛋白時，T細胞第四因子下游基因的調控也跟著改變，包括有cyclinD1和Hath-1基因，同時造成人類大腸癌細胞生長週期G1時期的停頓。除此之外，在人類大腸腺癌病人的檢體中，比較該病人癌化的與正常的大腸配對組織，Daxx蛋白的表現則有明顯減少的現象。綜合以上的發現推論Daxx蛋白可能透過與T細胞第四因子的結合，繼而調控人類細胞的生長週期，以達到細胞增生與細胞分化的生理目的。 另外，當共同表現Daxx蛋白時，可以透過SDS-PAGE的分離，觀察到另一個較小型態的T細胞第四因子蛋白的存在，並且隨著Daxx蛋白的表現劑量或時間有專一的相關聯性。T細胞第四因子的轉譯活性也可以被SUMO-1以及PIAS3加強，不過這樣的轉譯加強效果仍然可以被Daxx蛋白所抑制。以上的研究發現與推論，除了可以進一步勾勒出T細胞第四因子與Daxx蛋白結合時對人類生理功能的影響，同時也可以提供創新的思考模式，達到癌症治療的目的。; Recently, it has been reported that the nuclei of colon carcinoma cell lines contain constitutively active Tcf4 / b-catenin complexes as a direct consequence of either loss of function of the tumor suppressor gene APC or gain of function mutations in b-catenin itself. This is believed to result in the uncontrolled transcription of TCF target genes, leading to transformation of colon epithelial cells and initiation of polyp formation. Regulation of the transcriptional activity of b-catenin / Tcf4 has important implications for embryonic development as well as for carcinogenesis in the intestinal epithelium. Prior to discuss the potential role for LEF / TCF transcription factors in cancer, it is important to outline the mechanism by which they have been proposed to operate. Although LEF / TCF transcription factors bind directly to DNA through their HMG domains, they are incapable of independently activating gene transcription. On the other hand, most experimental data support the view that TCF is a repressor when Wnt does not convert it into an activator. TCF can repress target genes as well as activate those same target genes in cells instructed to change developmental fate, there are several mechanisms by which this switch is achieved. We are interested in the possibility of the bridging protein that interacts with TCF during the regulation of transcriptional activity. Because of its predominant expression in the human colonic epithelium, the full-length Tcf4 is used as bait in yeast two-hybridization, and Daxx is isolated. Daxx, a human cell death associated protein, has been reported to mediate the Fas / JNK-dependent signals in the cytoplasm. However, several lines of evidence have suggested that Daxx is mainly located in the nucleus and functions as a transcriptional regulator. Co-immunoprecipitation in HEK-293T cells and yeast two-hybrid screen in Y190 cells are performed to identify the interaction between Tcf4 and Daxx, and co-immunoprecipitation is also used to map the binding regions of Tcf4. In the nucleus, Daxx reduces DNA binding activity of Tcf4 and represses Tcf4 transcriptional activity. Overexpression of Daxx alteres expression of genes downstream of Tcf4, including cyclin D1 and Hath-1, and induces G1 phase arrest in colon cancer cells. Besides, a reduction in Daxx protein expression is observed in colon adenocarcinoma tissue when compared with normal colon tissue. These findings suggest a possible physiological function of Daxx, via interaction with Tcf4, to regulate cell cycle progression, and hence cell proliferation and differentiation. Furthermore, a small form of Tcf4 is observed specifically in SDS-PAGE when Daxx is co-expressed in a dose- and time-dependent manner. Moreover, transcriptional activity of Tcf4 is enhanced by SUMO-1 and PIAS3, but this transactivation still can be repressed by Daxx. Taken together, these findings not only outline the functional link between Tcf4 and Daxx, but also provide a new idea to develop novel cancer therapies. Sun, 01 Jan 2006 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31938 2006-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33199 標題: Ｃ型肝炎病毒蛋白對細胞內甲型干擾素反應可能之影響; The Possible Effects of HCV Proteins on Cellular IFN-alpha Responses 作者: Tsai-Yi Lu; 呂采宜 摘要: C型肝炎病毒（HCV）的感染已經成為公共衛生以及傳染病學界的重要課題之一。根據世界衛生組織的統計，全世界約有百分之三的人為HCV的帶原者，其中高達百分之八十的人在二十到三十年之後會發生肝硬化，甚至最後死於HCV所引起的肝癌。目前僅有長效型的甲型干擾素（IFN-α）合併抗病毒藥物Ribavirin可治療HCV的感染，但是成效最多只有百分之五十至六十，而且隨著病毒基因型的不同也會有所差異。本研究主要從兩方面來探討造成HCV無法被IFN-α清除的分子機制。首先，我們確認HCV的NS3/4A、NS4B和NS5A蛋白質具有抑制IFN-α對下游JAK-STAT signaling活化的能力。在檢驗每個活化過程的步驟之後，發現NS4B可以阻止ISGF3 complex和DNA的結合，因而導致ISRE主導的基因轉錄無法被啟動。另一方面，我們也重新檢視core在ISRE活化過程當中的影響。在七個從病人血清裡分離出來、分屬基因型1b和2a的core clone當中，每個clone對於ISRE的活化和下游ISG的表現都有不同程度的影響。於是我們比對這七個core的胺基酸序列，並推測某些胺基酸的改變是造成此現象的原因。根據以上的結果，我們認為更多關於NS4B對抗IFN-α訊息傳遞所使用機制的研究，將有助於開發對抗HCV感染的新方法；同時我們也要指出，研究core胺基酸序列的改變與其如何影響JAK-STAT signaling的關係，將有助於釐清core在HCV對抗IFN-α時所扮演的角色。; Hepatitis C virus (HCV) infection is one of the major problems of public health worldwide. Approximate 3% of the world’s population are chronically infected and may gradually develop chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (HCC) in the subsequent 20 to 30 years. IFN-alpha is one of the cytokines produced by most animal cells to resist viral replication and is by far the most standard therapy for HCV infection. However, the overall achievement of eliminating HCV only reaches 50-60% and is significantly altered by the virus genotypes. Here we investigated the underlying mechanisms from two aspects. First, we investigate the mechanisms that NS3/4A, NS4B, or NS5A used to inhibit the IFN-alpha-induced responses. The signaling of ISRE activation was examined step by step. It was found that NS4B significantly reduced the DNA binding ability of ISGF3. Second, we examined the effects of core on the ISRE activity using seven clones derived from patient sera infected by HCV genotype 1b or 2a. Distinct properties on ISRE activity and expression of ISGs were demonstrated clone by clone, and some putative amino acid changes were proposed to be responsible for these obscure results. We thus suggested that HCV NS4B deserved more attention for the development of new anti-HCV strategies and when attempting on unraveling the character of core, one should beware of the influence caused by every single amino acid substitutions. Sun, 01 Jan 2006 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33199 2006-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64713 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. Sun, 01 Jan 2012 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64713 2012-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44651 標題: 龐貝氏症新生兒篩檢偽陽性事件對父母擔任親職之長期影響; Long-term effect of false-positive Pompe disease newborn screening on parenting 作者: Hsiao-Ling Chen; 陳曉玲 摘要: 新生兒篩檢是一種 「以區域性人口族群」為背景的公共衛生政策，目的是降低某些先天代謝異常疾病的殘障率、疾病嚴重度或是死亡率。近年來，由於人類基因圖譜製作完成，衍生了許多新興科技、技術的發展與應用，也因此帶動了新生兒篩檢的發展，陸陸續續增加了新的疾病項目，龐貝氏症便是其中之一。本研究的目的是探討在某大醫院遺傳諮詢中心歷經龐貝氏症新生兒篩檢複檢及疾病確認事件後，篩檢結果為偽陽性之個案父母，其長期擔任親職時是否受到該複檢及疾病確認事件之影響。一共收集了研究組「有歷經龐貝氏症篩檢偽陽性的事件」之家長所填具的有效問卷19份，對照組「沒有歷經龐貝氏症篩檢偽陽性的事件」之家長所填具的有效問卷32份。 研究的結果發現：研究組與對照組在「疾病不確定感」、「親職壓力」、「親職勝任感」三大主變項總分的比較中，在「疾病不確定感」(P=0.04)主變項達到顯著差異，對照組得分較高，表示研究組的疾病不確定感高於對照組。在「缺乏資訊」、「不明確」、「模糊不清」、「無法預測」、「親職愁苦」、「互動失調」、「困難兒童」、「效能」、「挫折」九項次級變項的比較中，在「缺乏資訊」(P=0.003)與「不明確」(P=0.006)次級變項達到顯著差異，且對照組得分高於研究組，顯示研究組在「缺乏資訊」與「不明確」向度上的疾病不確定感比對照組來的高。另外，將研究組分為「擔心」與「不擔心」篩檢結果2組，再作比較，結果發現「擔心」與「不擔心」在三大主變項的比較中，均未達顯著差異，但是在「親職壓力」主變項中有達到顯著差異的趨勢(P=0.055)；九項次級變項的比較中，則是在「互動失調」(P=0.008)次級變項中達到顯著差異，且在「親職愁苦」(P=0.057)次級變項中，有達到顯著差異的趨勢，且都是「不擔心」得分較高。此結果顯示「有歷經龐貝氏症篩檢偽陽性的事件」且在篩檢複檢與疾病確認過程中表達「擔心」的父母，在「互動失調」向度上的親職壓力，會比表達「不擔心」的父母高。 將研究組與對照組之間在基本資料上達顯著差異的「家庭月收入」與「孩童年齡」項目，以Pearson’s Correlation分析所有的樣本，檢驗這2個項目與九項次級變項得分以及三大變項得分之間的的相關性；結果發現「孩童年齡」與九項次級變項之間皆未達顯著相關，只有與「缺乏資訊」(相關係數=-0.284)次級變項得分之間有達到顯著負相關的趨勢。「家庭月收入」則「困難兒童」(相關係數=+0.292)次級變項得分呈現顯著正相關，表示在本研究樣本中，「家庭月收入」越高，「困難兒童」次級變項的得分也會越高，也就是在「困難兒童」向度的親職壓力會越低。三大變項之間皆呈現顯著正相關，相關係數分列如下： *「疾病不確定感」與「親職壓力」之間相關係數為+0.456，「疾病不確定感」得分越高，「親職壓力」得分也越高，也就是不確定感越低，親職壓力越低。 *「疾病不確定感」與「親職勝任感」之間相關係數為+0.519，「疾病不確定感」得分越高，「親職勝任感」得分也越高，也就是不確定感越低，親職勝任感越高。 *「親職壓力」與「親職勝任感」之間相關係數為+0.729，「親職壓力」得分越高，「親職勝任感」得分也越高，也就是親職壓力越低，親職勝任感越高。 顯示在本研究樣本中以「親職壓力」與「親職勝任感」之間的相關性最強，「疾病不確定感」與「親職勝任感」之間的相關性次之，「疾病不確定感」與「親職壓力」之間的相關性最弱。本研究中發現龐貝氏症新生兒篩檢偽陽性結果，長期來說並不會造成家長擔任親職時的親職壓力上升，但由於研究組與對照組在「疾病不確定感」主變項的比較中達到顯著差異，且本研究中也發現「疾病不確定感」、「親職壓力」、「親職勝任感」的得分有顯著正相關的關係；若是存有過高的不確定感，可能會造成親職勝任感降低與親職壓力變高。 這些歷經篩檢偽陽性事件的家長們要面對的是孩子可能患有重大疾病的疑慮，以及後續是否進行基因檢測的選擇，因此醫療照護體系中的訊息傳達與關係建立可能還需要再特別加以關注，所以提供相關的遺傳諮詢服務是有必要的。; Newborn screening is a public health policy aims at reducing disability, severity of illness, or mortality of inborn errors of metabolism. After the success of the human genome project a few years ago, new science and technology allow more diseases to be screened at the newborn stage, like Pompe disease. The purpose of this study is to explore long-term effect of Pompe disease newborn screening on parenting. We conducted 19 questionnaires on the parents whose babies have experienced a false-positive Pompe disease screening (the study group). We also conducted 32 questionnaires on parents whose babies have a negative Pompe disease screening (the control group). We found that the total scores of 'Parenting Stress', and 'Parenting Sense of Competence' are not significantly different between the study group and the control group. There is significantly different between the study group and the control group in the total score of 'Uncertainty of Illness'. Among the nine sub-variables ('Lack of Information' , ' Lack of Clarity', 'Multiattributed Ambiguity', 'Unpredictability', 'Parental distress', ' Parent-Child dysfunctional interaction', 'Difficult Child', 'Efficiency', and 'Frustration'), we found that the scores of 'Lack of Information' and ' Lack of Clarity' sub-variable are higher in the control group than in the study group. This suggests that the study group is more uncertain about illness than the control group. This result is not related to whether the parents are 'worried' or 'not worried' about the screening result. But these parents, who expressed 'worried' about the screening result during false positive event, are perceiving more parenting stress in ' Parent-Child dysfunctional interaction' dimension than expressed 'not worried' about the screening result. One problem of the study is that the study group and the control group difference in both 'family income' and 'child age'. We analyze these two items in respective to either the three main variables or the nine sub-variables by Pearson's Correlation. We find that 'Family income' is positive correlated with 'Difficult Child'. Among the three main variables, each one is significantly positively correlated to the other two variables, respectively. In conclusion, a false positive Pompe disease newborn screening does not affect parenting significantly in 'Parenting Stress' and 'Parenting Sense of Competence', but the significantly different in 'Lack of Clarity' needs an attention. We need to be careful about delivering messages of newborn screening. Test instruction is very important, because the parents may face the fact having their children suffered from major illness. Service of genetic counseling is certainly needed. Fri, 01 Jan 2010 00:00:00 GMT http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44651 2010-01-01T00:00:00Z