http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/151 2026-03-12T07:40:07Z 2026-03-12T07:40:07Z Wei-Ming Chen 陳薇名 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76904 2021-07-10T21:40:03Z 2020-01-01T00:00:00Z

標題: 高脂飲食促進肺腺癌腫瘤生長機制探討; Investigation of Enhanced Lung Adenocarcinoma Progression Induced by High-Fat Diet 作者: Wei-Ming Chen; 陳薇名 摘要: 飲食習慣與癌症的發生明顯相關，據估計約30%的癌症病例與飲食有關。某些具抗癌效果的營養素被發現可抑制癌細胞生長的路徑，相反的部分脂質、低密度膽固醇及脂肪分泌的激素會促進癌細胞生長，於大腸癌、乳癌、前列腺癌等曾被報導，但在肺癌的探討相當有限且機制尚不明瞭。我們利用基因轉殖小鼠以藥物促使突變型表皮生長因子受體L858R表現作為肺腺癌動物模式，小鼠另外餵食高脂飲食及正常飲食以比較肺部腫瘤的生長情形，以及後續蛋白及基因的表現分析。結果顯示餵食高脂飲食的肺癌小鼠其腫瘤體積較大，Ki67的基因表現也顯著上升。利用RNA定序分析全基因表現發現高脂及正常飲食的肺腫瘤小鼠之基因表現主要差異在細胞分裂的功能，經qPCR確認ROS1基因表現在高脂飲食的肺腫瘤小鼠中顯著上升，且與腫瘤大小有高度正相關 (R=0.7058, p=0.0033)，然而ROS1蛋白磷酸化及其下游訊號無明顯差異。此外，FOXM1基因表現量亦在高脂飲食的肺腫瘤顯著上升，其功能已有促進腫瘤生長的報導。另一方面，在餵食野生種小鼠高脂飲食可使鼠蹊部白脂肪的TIMP1、pentraxin3和IL-6分泌量顯著上升，亦與RNA定序結果不謀而合。Kaplan-Meier 生存分析顯示高表現TIMP1或IL-6的肺腺癌患者其總存活期顯著下降。未來可用重組蛋白或抑制劑測試癌細胞的生長變化，以期能找出高脂飲食促使肺腫瘤生長的關鍵並進而應用於臨床。; Diet is attributed to about 30% of risk factors of cancers. Some nutrients with anti-tumor effect is also known for inhibition of signaling pathway related to tumor formation. In contrast, specific lipid uptake, low-density lipoprotein cholesterol and cytokine produced by adipose tissue can induce cancer cells proliferation and reported to correlate with several cancers. However, investigation of lung cancer and diet are limited and the mechanism is not clarified yet. We have bred inducible transgenic mice with spontaneous lung cancer formation driven by mutant EGFRL858R for animal model of lung adenocarcinoma. Lung tissues were harvested for hematoxylin and eosin (HE) stain, immunohistochemical (IHC) stain, western blotting and RNA for whole-genome transcriptomic analysis. Mice fed with high-fat diet and tumor induction (LC-HFD group) had larger tumor burden than regular-diet group (LC-RD) in observation of HE, IHC staining for Ki67 and lung weight. Gene set enrichment analysis of RNA sequencing revealed that the gene expression related to the function of cell division was more enhanced in LC-HFD than LC-RD. In addition, expression of ROS1 gene in LC-HFD was also increased significantly and highly correlated to tumor burden. However, there was no obvious phosphorylated ROS1 protein in western blotting and activation of downstream signals. The sequencing result also indicated that FOXM1 expression increased in LC-HFD, a transcription factor that has been known for promoting tumor growth. Tumor progression induced by high-fat diet may be triggered by FOXM1 rather than ROS1. On the other hand, TIMP1, pentraxin3, and IL-6 expression from inguinal white adipose tissue (iWAT) of high-fat diet treated wild-type mice were significantly upregulated, complying with the result of iWAT RNA sequencing. Kaplan-Meier plot showed high expression of TIMP1 or IL-6 was associated with lower overall survival in patients with lung adenocarcinoma. In order to find out the key leads high-fat diet promoting tumor progression, it is suggested to utilize recombinant protein or inhibitor for in vitro and in vivo assay to clarify the underlying mechanism in future.

2020-01-01T00:00:00Z Tzu-Hsuan Chuang 莊子璇 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38629 2021-06-13T16:39:48Z 2011-01-01T00:00:00Z

標題: 骨髓系增生疾病病人之端粒長度之研究：俱潛力之診斷標的; Study on Telomere Length in Patients with Myeloproliferative Neoplasms: A Potential Diagnostic Marker 作者: Tzu-Hsuan Chuang; 莊子璇 摘要: 骨髓系增生疾病(Myeloproliferative neoplasms, MPNs)是骨髓系幹細胞發生異常變化使得下游的血液細胞發生過度增生所造成的一種疾病，典型的骨髓增生性疾病病人可分為真性紅血球增多症、血小板增多症和原發性骨髓纖維症。近年來有研究發現在細胞中負責保護染色體末端的端粒，其長度在骨髓增生性疾病病人中有明顯縮短的趨勢。我們藉由一對能結合至端粒重複序列的引子與一對能結合至單一基因(single copy gene)的引子，利用即時監控聚合酶連鎖反應的原理來測量各檢體的相對端粒長度--相對端粒長度(Relative telomere length)正比於端粒產物量(T)比上單一基因產物量(S)之比例(T/S ratio)。 我們分析63 位被測得有高於正常人血球數目的病人其端粒長度並與255 位測有正常血球數目的人做比較，再將此端粒長度的分析結果與其他骨髓增生性疾病突變檢測結果綜合評估。因為在正常人群組中，端粒長度會受個人的性別與年齡影響，因此這些因素在病人中亦被分別分析。 在63 位被測得有高於正常人血球數目的病人中有35 位為JAK2 V617F 陽性的病人。其中27 位V617F 陽性病人的端粒長度較同齡正常人有明顯縮短之現象。此外此端粒長度縮短的現象只有在病人的多核細胞檢體中被發現，在其單核細胞檢體中則否。而在不同性別的V617F 陽性病人比較中，女性病人與男性病人的端粒長度則沒有顯著的差異。在63 位被測得有高於正常人血球數目的病人中剩下的28 位為JAK2 V617F 陰性的病人，藉由甲基特異化聚合酶連鎖反應 (Methylation-specific PCR assay)的結果我們將V617F 陰性的病人分為在JAK2的抑制子SOCS3 promoter 有部份甲基化及無甲基化組。而在SOCS3 promoter有部份甲基化的四位病人中，都可發現其端粒長度都有較同齡正常人下降的表現。 因此實驗認為端粒長度分析可作為檢測JAK2V617F 陰性骨髓增生性疾病中於JAK2 的抑制子SOCS3 promoter 有部份甲基化的病人之另一項標的。; Myeloproliferative neoplasms (MPNs) are caused by several disorders of myeloid clonal stem cells and these diseases are characterized by chronic proliferation of hematopoietic precursors. There are three major types of classic myeloproliferative neoplasms: polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), and JAK2V617F is the most common biomarker to screen the patients with classic MPNs. Recent studies have reported that telomeres, which are complexes of tandem repeats and able to protect the chromosomes against homologous recombination and non-homologous end joining during cell division, showed significant shortening in patients with MPNs. In this study, the quantitative-PCR assay measured the relative telomere length of 63 studied patients with abnormal increased leukocyte count, or increased platelet count, or increased hemoglobin level; and then the results of the patients was compared with 255 normal controls which have normal complete blood counts (the relative telomere length was the ratio of telomere product to single-copy gene product, T/S). Due to the telomere length was affected by different genders and ages in the normal control cohort, the comparisons of the patients between different genders and ages were processed separately. In the JAK2V617F-positive patient cohort, the results of paired comparisons showed that telomere lengths were remarkably shorter in 27 of 35 patients (27/35) than the age-matched normal controls. The telomere length shortening was observed mainly in the granulocytes but not in the mononuclear cells. Furthermore, no significant differences of telomere shortening were observed between female and maleJAK2V617F-positive patients. In addition, the mutation burden of JAK2V617F displayed no obvious correlation with telomere length shortening in the JAK2V617F-positive patient cohort. In the JAK2V617F-negative patients, abnormal SOCS3 promoter methylation pattern was found in four patients who had significantly shorter telomere lengths than their age-matched normal controls. SOCS3 promoter methylation has been reported among the patients with PMF in the previous reports, so these four SOCS3-positive patients with shorter telomere lengths could have primary myelofibrosis. In conclusion, this data suggested that telomere length analyses may support the diagnoses in patients with SOCS3 promoter methylation but without JAK2V617F.

2011-01-01T00:00:00Z Pi-Han Lin 林必涵 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9480 2021-05-20T20:24:37Z 2011-01-01T00:00:00Z

標題: 香豆素衍生物BPRHIV001藉由Akt途徑抑制第一型人類免疫缺乏病毒Tat蛋白質調節之轉錄活性; Inhibition of HIV-1 Tat-mediated transcription by a coumarin derivative BPRHIV001 through Akt pathway 作者: Pi-Han Lin; 林必涵 摘要: 第一型人類免疫缺乏病毒(Human immunodeficiency virus type 1, HIV-1)的Tat調節蛋白質是病毒轉錄RNA所必需。因此，Tat功能受影響時，病毒的複製將會被抑制。先前我們設計一個可偵測Tat功能的細胞篩選平台，來篩選抑制HIV-1的新興化合物。經此平台篩選了292個香豆素衍生物，其中BPRHIV001在奈莫耳濃度（nanomolar concentration）即具有抑制Tat轉錄活化的活性。進一步實驗發現，BPRHIV001不是透過影響Tat的生成以及Tat轉錄活化相關蛋白質複合物- Tat/P-TEFb組成，來抑制其對RNA第二型聚合酶（RNAPII）的活化作用。因此我們想探究BPRHIV001是否透過調控Tat轉譯後的修飾來影響其功能。結果發現，加入BPRHIV001後，細胞內可以乙醯化Tat並且幫助其離開TAR RNA的p300蛋白質量明顯減少，但mRNA量並沒有受到影響。因為磷酸化的Akt可穩定p300蛋白質，減緩其衰減，我們接著發現Akt及其活化分子PDPK1的磷酸化都在BPRHIV001的存在下降低許多。我們進行蛋白質嵌合分析(Docking analysis)來探討BPRHIV001與PDPK1之間的關係，結果顯示BPRHIV001可能藉由變構效應(allosteric effect)與PDPK1交互作用進而抑制PDPK1的磷酸化使其活性降低。最後in vitro實驗發現，BPRHIV001可有效抑制HIV-1的複製，其50%有效濃度（50% inhibitory concentration；IC50）為1.3 nM。BPRHIV001也可與現行使用的反轉錄酶抑制劑azidothymidine (AZT) 以及efavirenz (EFV)有強烈的加成效應。 綜觀以上結果，BPRHIV001可能透過干擾PI3K/Akt 途徑，來抑制HIV-1 Tat蛋白質所調控的轉錄活化活性。由於其與現行使用的抗病毒藥物具有良好的加成作用，我們認為BPRHIV001可望發展成為抗HIV-1感染的新興治療藥物的前導化合物。; The HIV-1-encoded RNA-binding protein Tat is known to play an essential role in viral gene expression. In the search of novel compounds in inhibiting Tat transactivity, BPRHIV001 was identified after screening of 292 coumarin-derivatives. BPRHIV001 was shown to significantly inhibit Tat-mediated transactivation at nanomolar range. BPRHIV001 is likely to exert its effects at the stage after initiation of RNAPII elongation since Tat protein expression and the assembly of Tat/P-TEFb complex which is essential for processivity of RNAPII elongation complex, remained unchanged. Next, the level of p300 was determined since acetylation of Tat by p300/CBP could facilitate the dissociation of Tat from TAR and subsequent recycling of Tat. The p300 protein level was reduced in the presence of BPRHIV001, while the p300 mRNA level was unaffected. The reduced p300 protein level was possibly resulted from reduced stability of p300 since the level of phosphorylated Akt, which was shown to be closely related with p300 stability, decreased in the presence of BPRHIV001. A concordant reduction of phosphorylated PDPK1, a well-known Akt activator was also observed. Furthermore, the docking analysis revealed that the reduced PDPK1 phosphorylation is likely resulted from the allosteric effect of interaction between BPRHIV001 and PDPK1. Finally, BPRHIV001 was shown to effectively inhibit HIV-1 replication in vitro and the 50% inhibitory concentration (IC50) of BPRHIV001 against HIV-1 was 1.3 nM. BPRHIV001 also had strong synergistic effect with current reverse transcriptase inhibitor azidothymidine (AZT) and efavirenz (EFV). Above all, BPRHIV001 was shown to have strong inhibitory effect on HIV-1 Tat-mediated transactivation, possibly through repressing the PI3K/Akt pathway.With strong synergistic effects with current reverse transcriptase inhibitors, BPRHIV001 has the potential to become a promising lead compound for the development of novel therapeutic agent against HIV-1 infection.

2011-01-01T00:00:00Z Yi-Jyun Lin 林怡君 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72974 2021-06-17T07:12:22Z 2019-01-01T00:00:00Z

標題: 非血檢體分離之甲氧西林敏感金黃色葡萄球菌之抗紅黴素基因分析; Erythromycin resistance genes in methicillin-susceptible Staphylococcus aureus isolated from non-blood specimens 作者: Yi-Jyun Lin; 林怡君 摘要: 金黃色葡萄球菌的抗藥性，對世界各地來說都是一項棘手的危機，紅黴素是臨床上常用來治療革蘭氏陽性球菌的藥物，紅黴素抗藥菌株問題也相當嚴重，因此，本研究選擇目前較少被注意到，但仍具有臨床重要性的非血來源MSSA，進行抗紅黴素菌株的相關探討。菌株收集2017年9月到2018年2月，經台大醫院細菌室自非血液檢體培養，並鑑定為抗紅黴素MSSA的分離株，總共有209株。利用PCR分析抗藥基因，以及用抗生素敏感性試驗確認抗藥表現型和紅黴素MIC後，發現帶msrA/B基因的有124株，佔總數的59%，其MIC值普遍低於帶erm基因的菌株，而M表現型則有128株(61%)。另以spa typing、PFGE和MLST分析實驗菌株的演化關係，發現最主要的pulsotype包含104株菌，屬於ST15，他們的spa type則是占最大宗的t084和t085，以上種種皆透露出有clonal spreading的狀況，在試圖尋找造成ST15具備感染優勢的因素後，發現可能與毒力因子相關，但仍有待深究。此外，本研究亦針對ermT基因做進一步鑽研，2017到2018半年間共收集到四株帶有ermT基因的MSSA，皆以染色體DNA為攜帶方式，且四株的sequence types都是ST398，此ST是目前帶有ermT的主流。 其中T17.56、T17.83、T18.05三株攜帶ermT的結構與實驗室於2011年發現的NTUH-8300相比，僅在rep基因周圍有些微差別，整體來說，皆為一段和pUR3912質體高度相似的序列，藉由IS431插入在細菌染色體DNA的transposase基因中；而T18.58帶ermT的完整結構尚未明瞭，只知道與目前已知者有很大的不同，包含一段同時比對到ST398質體和ST9染色體的序列，推測由外來DNA插入。除此之外，也承襲過去實驗室成員未完成的NTUH15-4 ermT結構解析，結果顯示由包含ermT基因的pUR3912質體，和pT45質體相鄰插入染色體DNA的pepT_2基因中，兩質體之間還存在一個抗藥基因aadD，而兩質體外側的序列比對到ST9 CC9金黃色葡萄球菌。特別的是，NTUH15-4為一株MSSA ST834-CC9，目前國內外尚未有ST834攜帶ermT基因的報導。; Drug resistance in Staphylococcus aureus is a major problem worldwide. Erythromycin plays an important therapeutic role in Staphylococcal infections. However, overuse of antibiotics for a long time led to resistant strains development. In consideration of the crisis, we performed an epidemiology investigation of MSSA isolated from non-blood specimens in northern Taiwan. Although the researches on MSSA were rarer than those on MRSA, we should still pay attention to MSSA due to its increasing rate of resistance. In the present study, a total of 209 non-blood isolates of erythromycin-resistant MSSA were collected from National Taiwan University Hospital from September 2017 to February 2018. After screening the erythromycin resistance genes by PCR and determining the MLSB resistance phenotypes by means of double disk diffusion assay, we found 124 (59%) isolates harbored msrA/B gene and M phenotype was predominant (128 of 209 isolates). We used PFGE, spa typing and MLST to analyze the molecular evolution of S.aureus. 104 (50%) isolates were grouped into the same pulsotype and typed as ST15-t084/t085. The results indicated clonal spreading during this period. The advantage of ST15 MSSA may be related to virulence factors. On the other hand, we also focused on one of the erythromycin resistance determinant, ermT. Four MSSA ST398 carried ermT gene. Three of them had the gene elements which were highly similar to NTUH-8300, a MSSA isolated from NTUH in 2011, except the rep gene region. This kind of element consisted of pUR3912 plasmid integrating into a transposon in chromosomal DNA. The other isolate, T18.58, carried ermT gene with another structure. It appeared that a plasmid inserted into chromosome DNA, but we haven’t identified it totally yet. Furthermore, our team found ermT gene in ST834-CC9 NTUH15-4 in 2015. Sequencing of the ermT and flanking regions in NTUH15-4 revealed that the ermT was located in chromosome. The element contained fragments from two plasmids, pUR3912 and pT45, and a tobramycin resistance gene, aadD. The integrated site is comparable to the pepT-2 gene in ST9 S. aureus.

2019-01-01T00:00:00Z