http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80 2026-04-13T06:36:04Z 2026-04-13T06:36:04Z Chuan-Chuan Lin 林嫥嫥 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6273 2021-05-16T16:24:44Z 2013-01-01T00:00:00Z

標題: 黃麴毒素B1對豬免疫細胞及豬第二型環狀病毒細胞內複製的影響; The Effect of Aflatoxin B1 (AFB1) on Swine Immunocytes and Intracellular Porcine Circovirus Type 2 (PCV2) Replication 作者: Chuan-Chuan Lin; 林嫥嫥 摘要: 黃麴毒素B1(AFB1)是常見於亞熱帶及熱帶地區飼料原料穀物的黴菌毒素，豬隻可能因攝食到黴菌汙染的飼料而接觸AFB1，由於長期接觸低劑量AFB1會影響動物的免疫系統，因此AFB1可能成為豬隻產生豬環狀病毒相關症(PCVAD)的環境因子。本研究自無臨床症狀的豬第二型環狀病毒(PCV2)感染豬隻分離周邊血液單核細胞(PBMCs)，並將細胞進一步分化為單核球來源的樹突細胞(MoDCs)以做為抗原呈獻細胞(APCs)的研究模式，檢測豬免疫細胞於in vitro條件下與AFB1共同後培養，對其細胞功能及PCV2複製的影響。在對免疫細胞功能影響的結果顯示，10 μg/ml 高濃度AFB1處理72小時會減少PBMCs受到致裂原Con A刺激後之細胞存活率及細胞增殖能力；10 μg/ml高濃度AFB1處理48小時會使MoDCs變小、變圓，並失去樹突狀結構，並會降低MoDCs的抗原攝取與處理能力；10 μg/ml高濃度AFB1處理24小時後會增加MoDCs的細胞表面抗原CD40、CD83及CD86，以及免疫抑制性細胞激素IL-10的 mRNA表現量；0.001 μg/ml 低濃度AFB1則在處理後6小時造成MoDCs的CD40及CD83 mRNA表現量上升。在對PCV2複製的影響上，PBMCs受到72小時致裂原Con A的活化下，雖然10 μg/ml AFB1處理並不影響整個處理單位內細胞中的總病毒量，但卻可能增加單位細胞內所含的PCV2病毒量；而MoDCs經過10 μg/ml AFB1處理72小時後，也有處理單位內細胞量減少但細胞中總病毒量與對照組含量相近的現象。本研究結果顯示10 μg/ml 高濃度AFB1有機會增加MoDCs及Con A活化之PBMCs單位細胞內的PCV2病毒複製、傷害細胞媒介性免疫中的APCs功能及細胞增殖能力，並與PCV2同樣能刺激細胞的IL-10 mRNA表現而造成免疫抑制，故可能促使攝食到飼料被高量AFB1汙染的PCV2次臨床感染豬隻發展為PCVAD患豬。; Aflatoxin B1 (AFB1) is a mycotoxin commonly found in cereal grain and related products in subtropical and tropical regions, and pigs may expose to this toxin through contaminated feed. Chronic, low-dose exposure of AFB1 is known to affect the immunity, and may be a potential environmental factor in association with porcine circovirus type 2 (PCV2) in the induction of porcine circovirus-associated disease (PCVAD). In this study, peripheral blood mononuclear cells (PBMCs) were isolated from healthy subclinically PCV2-infected pigs and further differentiated to monocyte-derived dendritic cells (MoDCs) as an antigen-presenting cells (APCs) model to investigate the changes in immunocyte functions and PCV2 replication after in vitro AFB1 exposure. The PBMCs viability and Con A-induced proliferation were impaired after treated with AFB1 at 10 μg/ml for 72 hours. The cell morphology of MoDCs became smaller or rounded, contained shorter dendrites, and lost the antigen uptaking and processing abilities after exposure to 10 μg/ml AFB1 for 48 hours. The mRNA expression levels of surface markers CD40, CD83, and CD86 and regulatory cytokine IL-10 increased in MoDCs after treated with 10 μg/ml AFB1 for 24 hours. A significantly up-regulated CD40 and CD83 mRNA levels were observed in MoDCs after treated with 0.001μg/ml AFB1 for 6 hours. The cell-based PCV2 load appeared to increase in 10 μg/ml AFB1-treated Con A co-treated PBMCs, although no significant difference was seen in the total copy number of PCV2 genome; similar increase in cell-based PCV2 load might also occurr in 10 μg/ml AFB1-treated MoDCs. These results indicate that a relatively higher concentration of AFB1 such as 10 μg/ml may have the potential to increase PCV2 replication in PBMCs and MoDCs, impair the APCs cell function and cell proliferation in cell-mediated immunity, and cause immunosuppression through enhancement of IL-10 mRNA expression. Thus, it is suggested that AFB1 may potentiate PCVAD development in subclinically PCV2 infected pigs through dietary exposure to AFB1.

2013-01-01T00:00:00Z Yen-Han Chen 陳彥涵 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80328 2022-11-24T03:04:34Z 2021-01-01T00:00:00Z

標題: 鳥類腦乙型類澱粉沉著症回溯性研究; A Retrospective Survey of Cerebral Beta-Amyloid Accumulation in Avian Species 作者: Yen-Han Chen; 陳彥涵 摘要: "阿茲海默症（Alzheimer’s Disease, AD）是人類老年醫學造成「失智症」最重要的疾病，約佔失智症人口60-70%。本病是進行性的神經性病變，可引起程度不一的認知功能障礙，最重要的組織病變包含：(1) 細胞外可見乙型類澱粉蛋白（Beta-amyloid）沉著，形成類澱粉斑塊（Aß plaques, senile plaques）或出現腦部類澱粉血管病變（Cerebral amyloid angiopathy, CAA）；(2) 細胞內過磷化tau蛋白（hp-tau）堆積，嚴重者可造成神經元壞死，形成神經元纖維纏結（Neurofibrillary tangles, NFTs）。至今，本病仍有部分致病機轉尚未全然解開。根據文獻，許多老年動物被發現有類似人類阿茲海默症的腦部病變，包含不同程度的乙型類澱粉蛋白（Beta-amyloid）沉積與神經元纖維纏結（NFTs）的神經元內堆積，包括靈長目、食肉目、鯨偶蹄目、囓齒目、兔型目、馬（奇蹄目）與其他海洋哺乳類，且於不同物種有不同的病變發展。不過，相關的研究在鳥類中仍非常少。本論文為老年鳥類乙型類澱粉蛋白（Beta-amyloid）沉著提供一個回溯性研究，並發現於鳥類可有三種不同型態的乙型類澱粉蛋白沉積，以及證實了腦血管乙型類澱粉沉著可於鳥類中被發現。本研究樣本共包含13個目，29個物種，共50隻個體，並以免疫組織化學染色進行四種（6E10、4G8、x-40、x-42）抗乙型類澱粉蛋白（Beta-amyloid）及一種（AT100）抗過磷酸化tau蛋白染色，偵測是否有AD相關之異常蛋白存在於鳥類腦組織中。研究結果發現，除一34歲白腹海鵰可在H E染色下看到明顯微小膠細胞增多以及聚集在神經纖維周圍之外，其他病例的腦組織皆未出現明顯組織病變。在IHC染色結果，本實驗首次發現了白腹海鵰腦組織病例可顯示出3+強陽性，且可見明顯類澱粉斑塊（Aß plaques）及多發局部的類澱粉血管病變（CAA），呈現diffuse to compact斑塊（Diffuse/compact plaque）。此外，另一熊鷹（赫氏角鷹，Nisaetus nipalensis）病例亦為首次發現，可見大腦及小腦腦膜血管出現明顯多發Aß陽性訊號。挑選幾個乙型類澱粉蛋白（Beta-amyloid）陽性樣本進一步以甲烯胺嗜銀染色（Methenamine silver，MS）染色及類澱粉樣染色（Congo Red stain），以確定類澱粉斑塊（Aß plaques）的結構存在。所有老年鳥類樣本經AT100染色後皆為陰性，未見明顯細胞內tau蛋白堆積的證據。而兩例IHC陽性病例，皆呈現類澱粉樣染色（Congo Red stain）陰性結果。此外，Aß (x)-40與42兩種抗體的染色，在本實驗結果討論中，被認為是於鳥類研究中較有意義的抗體。本研究提供關於老年鳥類腦部乙型類澱粉蛋白（Beta-amyloid）沉著的初步視野，並發現乙型類澱粉蛋白（Beta-amyloid）的異常堆積可能具有好發於鷹科海鵰屬（Haliaeetus spp.）或其他老年猛禽類的傾向，但相關疾病機轉仍需要更進一步的探究。據此，老年鳥類可能是自然發生類阿茲海默症腦部病變的動物，可出現與人類或其他哺乳動物類似乙型類澱粉蛋白（Beta-amyloid）的堆積，但沒有神經原內的異常tau蛋白形成。"

2021-01-01T00:00:00Z Jou-Fei Wu 吳柔霏 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79271 2022-11-23T08:57:06Z 2021-01-01T00:00:00Z

標題: 辨識葡萄糖調節蛋白78為豬流行性下痢病毒可能之細胞輔助受器蛋白; Identification of Glucose Regulated Protein 78 as a Potential Cellular (Co-) Receptor Protein for Porcine Epidemic Diarrhea Virus 作者: Jou-Fei Wu; 吳柔霏 摘要: 豬流行性下痢病毒（Porcine epidemic diarrhea virus; PEDV）屬於冠狀病毒屬中的甲型冠狀病毒，可引發具有高度傳染性的豬流行性下痢（Porcine epidemic diarrhea; PED），受其感染之仔豬會出現急性水漾腹瀉、嘔吐及脫水，最終導致死亡。PEDV之細胞受器最初被認為是豬丙胺酸胺肽酶（Porcine Aminopeptidase N; pAPN），但pAPN是否為PEDV細胞受器卻在近幾年受到質疑。在本研究中，我們利用免疫共同沈降與質譜分析技術，將PEDV棘狀蛋白（Spike; S）三聚體分別與新生仔豬之腸上皮細胞和非洲綠猴腎細胞（Vero-E6 cell）膜蛋白進行交互作用，以辨識PEDV之可能細胞受器。本研究發現在新生仔豬腸細胞與Vero-E6細胞膜蛋白中葡萄糖調節蛋白78（Glucose regulated protein 78; GRP78）可與PEDV S蛋白進行免疫沉降。為了更近一步探討GRP78在PEDV感染機制中所扮演的角色，我們轉染豬腸上皮細胞株 (Intestinal porcine epithelial cell line-1; IPEC-1)與豬睪丸細胞豬 (Swine testicular cell line; ST )使其可以穩定表現GRP78後，再以第七代PEDV-PT-52（PEDV-PT-P7)進行了感染試驗，並與未轉染之IPEC-1細胞、ST細胞與Vero-E6細胞進行比較。結果發現相對於病毒感染之Vero-E6細胞可見特徵性融合細胞形成之外，轉染GRP78之IPEC-1細胞與ST細胞以及未轉染之IPEC-1細胞與ST細胞均無法觀察到細胞病變作用（Cytopathic effect）。此外，除了Vero-E6 細胞株外，本實驗其他細胞株的上清液均無法檢測到病毒核酸。依據實驗結果推測，GRP78雖能與PEDV S進行鍵結，但並非扮演宿主細胞受器之角色，而GRP78是否能夠做為輔助細胞受器蛋白協助病毒的組裝或複製則需要後續實驗證明。

2021-01-01T00:00:00Z Chen-Hui Lu 陸辰惠 http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84515 2023-03-19T22:14:05Z 2022-01-01T00:00:00Z

標題: 貓注射部位肉瘤：轉錄體剖析及第二型環氧合酶所扮演之角色; Feline Injection Site Sarcoma: Transcriptome Profile and Role of Cyclooxygenase-2 作者: Chen-Hui Lu; 陸辰惠 摘要: 貓注射部位肉瘤（feline injection-site sarcomas, FISS）是種發生在貓的間質來源惡性腫瘤，其具有高度侵犯性且被認為與注射行為相關，但致病機轉尚未明瞭，目前最廣為接受的假說是，因注射相關創傷和異物性化學物質刺激引發慢性炎症，進而導致FISS的發生。由於慢性炎症能夠提供有利於腫瘤生長的微環境，故在多種腫瘤被認為是誘發腫瘤發生的潛在因子。為了瞭解FISS的腫瘤發生與尋找具有潛力的治療標靶，本研究選擇環氧合酶-2（cycloxygenase-2, COX-2）作為研究目標，並以FISS及正常組織的初代細胞進行COX-2抑制劑（robenacoxib)的體外試驗，評估COX-2抑制劑作為輔助治療藥物的可能性與研究COX-2在FISS腫瘤發生所扮演的角色。此外，本研究也利用次世代基因定序，經由大數據分析FISS與正常組織之基因表現差異，以尋找其他可能的腫瘤生成機制。結果顯示，本研究使用的FISS病例之石蠟包埋組織與初代細胞均可見不等程度的COX-2表現，COX-2抑制劑能夠抑制FISS初代細胞的細胞存活率、移行和細胞群落形成，以及誘發細胞凋亡，並且表現明顯的劑量依賴性，但不同細胞株的敏感性有所差異且不完全與COX-2的表現量相關。本研究顯示COX-2抑制劑可能具有作為FISS輔助治療藥物之潛力。在次世代定序方面，FISS初代細胞不僅在腫瘤途逕如Wnt、Ras以及Rap1的基因表現有顯著變化，炎症反應相關途徑如PI3K-Akt和MAPK亦可見顯著差異，故FISS的腫瘤發生可能與多種炎症反應途徑有關，其扮演的角色仍需要進行更深入的探討。; Feline injection-site sarcomas (FISSs) are highly invasive malignant mesenchymal neoplasm arising from the injection sites in cats. Although the tumorigenesis of FISSs is still uncertain, it is the consensus that FISS is associated with chronic inflammation caused by the irritation of injection-related trauma and foreign chemical substance. Owing to that chronic inflammation can provide a proper microenvironment for tumor development and thus has been known as one of the risk factors of tumorigenesis in many tumors. To investigate the tumorigenesis of FISS and screening potential therapeutic targets of FISS, cyclooxygenase-2 (COX-2), an inflammation enhancing enzyme, is selected as the target of the study and in vitro experiments using FISS- and normal tissue-derived primary cells and robenacoxib, a highly selective COX-2 inhibitor, were performed. In order to screening the potential pathways associated with FISS tumorigenesis, next generation sequencing (NGS) was conducted to compare the differences in gene expression between cells derived from FISS and normal tissue. The results demonstrated that the expression of COX-2 could be detected in formalin-fixed and paraffin embedded FISS tissues and FISS-derived primary cells. The cell viability, migration and colony formation of FISS-derived primary cells could be inhibited and the cell apoptosis could be enhanced by robenacoxib in a dose dependent manner. However, the susceptibility to robenacoxib varied in different lines of FISS primary cells and was not completely correlated with the expression of COX-2. The results of the study suggests that COX-2 inhibitor may have potential as an adjuvant treatment for FISSs. As for the results of NGS, significantly differentially expressed genes associated with not only the pathways in cancer, such as Wnt, Ras and Rap1 signaling pathway, but also pathway associated with inflammation, such as PI3K-Akt and MAPK signaling pathway were revealed. Therefore, the tumorigenesis of FISS might be corelated with multiple inflammatory pathways and their roles on FISS development still need further investigation.

2022-01-01T00:00:00Z