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系統識別號 U0026-1408201017375500
論文名稱(中文) 宿主免疫細胞與陰道鞭毛蟲交互作用研究-活性氧分子與細胞激素觀點
論文名稱(英文) The interaction between Trichomonas vaginalis and human HL-60 cell - Reactive oxygen species’ and cytokines’ point of views
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 98
學期 2
出版年 99
研究生(中文) 宋昀儒
研究生(英文) Yun-Ju Sung
學號 S4697408
學位類別 碩士
語文別 中文
論文頁數 79頁
口試委員 指導教授-辛致煒
口試委員-楊倍昌
召集委員-蘇霩靄
口試委員-鄧致剛
中文關鍵字 陰道鞭毛蟲  陰道鞭毛蟲症  嗜中性白血球  活性氧分子  細胞激素 
英文關鍵字 Trichomonas vaginalis  Trichomoniasis vaginalis  Neutrophil  Reactive oxygen species (ROS)  Cytokine 
學科別分類
中文摘要 陰道鞭毛蟲 (Trichomonas vaginalis) 是一種厭氧性的單細胞原生寄生蟲,感染所引起的陰道鞭毛蟲症 (trichomoniasis vaginalis) 是最常見的非病毒的性接觸傳染疾病。已知研究指出陰道鞭毛蟲的感染會引發感染部位的急性發炎傷害,伴隨著有許多陰道鞭毛蟲的存在、嗜中性白血球的浸潤以及上皮細胞的破壞。依據本實驗室先前的研究以及臨床的研究結果指出,嗜中性白血球的趨化激素 IL-8 可表現於陰道鞭毛蟲症患者之患處,以及與陰道鞭毛蟲共同培養的子宮頸上皮細胞上,但這些受到趨化作用而浸潤到感染處的嗜中性白血球是如何與陰道鞭毛蟲進行交互作用,這個部分仍不是非常清楚。在本篇研究中,利用嗜中性白血球的細胞株 HL-60 與陰道鞭毛蟲進行共同培養。我們發現越多的陰道鞭毛蟲存在,會造成 dHL-60 (differentiated HL-60) 產生內生性的活性氧分子 (reactive oxygen species, ROS) 越低,這是一個存在有感染程度以及時間性的調控關係。在 cytokine microarray 及 RT-PCR 的結果顯示,當陰道鞭毛蟲與 dHL-60 共同培養後,會引發細胞激素及趨化激素 IL-1β、TNF-α 及 IL-8 表現上升,ATP6AP1 則是有下降表現的現象。進一步,我們同樣利用 RT-PCR 的方式,觀察陰道鞭毛蟲的 oxygen scavenging system 的基因是否會與 dHL-60 共同培養後所產生的反應。我們發現在與 dHL-60共同培養的條件下,會造成不同程度 superoxide dismutase 6 (SOD6)、紅素氧還蛋白 rubrerythrin (RBR) 以及 thioredoxin peroxide (TrxP) 這些基因的表現。以上結果指出,陰道鞭毛蟲或許可以藉由降低宿主細胞活性氧分子的產生、調節細胞激素的表現,或者提升自身清除氧化物的能力來逃避先天性免疫反應的攻擊。
英文摘要 Trichomoniasis vaginalis, is one of the most common non-viral sexually transmitted diseases in human and caused by infection an anaerobic protist Trichomonas vaginalis. It is known that infection elicits a profuse, acute, inflammatory discharge containing many trichomonads, neutrophils infiltration and epithelial cells destruction. Although our and clinical studies showed chemoattractants of neutrophils, such as IL-8, are found in vitro culture between T. vaginalis and cervical epithelial cell or vaginal discharges of symptomatic trichomoniasis patients, not much concern about the interaction between infiltrated neutrophil and T. vaginalis. In this study, NBT assay and flow cytometric results show more T. vaginalis incubate with dHL-60 cells, that are neutrophil-like cell line, but less intracellular reactive oxygen species (ROS) generate from dHL-60 cells in a dose- and time-dependent manner. In the cytokine microarray and RT-PCR results, we find IL-1β and IL-8 and TNF-α of dHL-60 cells are up-regulated.and down-regulated ATP6AP1. Moreover, genes of trichomonad’s oxygen scavenge system such as superoxide dismutase 6 (SOD6), rubrerythrin (Rbr) and thioredoxin peroxidase (TrxP) are up-regulated while co-incubation with dHL-60. These results suggest that T. vaginalis may escape from innate immune response by decreasing ROS production, regulating pro-inflammatory cytokine expression and decreasing and increase self-oxygen detoxication mechanism.
論文目次 摘要 I
Abstract II
誌謝 III
目錄 V
表目錄 VIII
圖目錄 IX

第一章 緒論 1
第二章 實驗設計 9
第三章 材料與方法 11
3.1 HL-60 細胞生長培養基配製 11
3.2 HL-60 細胞培養 11
3.2.1 HL-60 細胞解凍 11
3.2.2 HL-60 細胞繼代培養 12
3.2.3 HL-60 細胞分化培養 12
3.2.4 HL-60 細胞保存 13
3.3 HL-60細胞分化測定 13
3.3.1 細胞大小分析 13
3.3.2 免疫螢光染色 (Immunofluorescence stain) 13
3.3.3 細胞週期分析 14
3.3.4 NBT 還原試驗 14
3.4 陰道鞭毛蟲生長培養基配製 15
3.5 陰道鞭毛蟲培養 15
3.5.1 陰道鞭毛蟲解凍 15
3.5.2 陰道鞭毛蟲繼代培養 16
3.5.3 陰道鞭毛蟲馴化 (domestication ) 16
3.5.4 陰道鞭毛蟲保存 16
3.6 陰道鞭毛蟲生長曲線 (growth curve) 與倍增時間 (doubling time) 16
3.7 已分化細胞 (dHL-60) 與陰道鞭毛蟲共同培養 17
3.8 dHL-60 活性氧分子分析 18
3.8.1 NBT 還原試驗 18
3.8.2 DCFH-DA assay 18
3.9 RNA 萃取 18
3.10 Real-time PCR 19
3.10.1 cDNA 合成 19
3.10.2 PCR 反應 19
3.10.3 PCR核酸產物電泳 20
3.10.4 Real-time PCR 反應條件與步驟 20
3.11 微陣列分析 (microarray analysis) 21
3.12 Reverse Transcriptase PCR (RT-PCR) 22
3.12.1 RT-PCR 反應步驟 22
3.12.2 RT-PCR 反應條件 22
3.12.3 PCR 核酸產物電泳 23
3.13 統計分析 24
第四章 結果 25
4.1 利用 DMSO 使 HL-60 細胞分化成類嗜中性球 (neutrophil-like cell) 25
4.1.1 細胞大小與表面蛋白 CD11b 的表現分析 25
4.1.2 細胞生長情形分析 25
4.1.3 超氧化離子 (superoxide) 產生能力分析 26
4.2 陰道鞭毛蟲馴化 (domestication) 後的生長情形 27
4.3 共同培養 (co-incubation) 系統建立 27
4.4 dHL-60 活性氧分子分析 28
4.4.1 dHL-60 胞內活性氧分子測定 28
4.4.2 dHL-60 胞外活性氧分子測定 29
4.5 陰道鞭毛蟲對 dHL-60 產生細胞激素 (cytokine) 的影響 30
4.6 生物氧化壓力在陰道鞭毛蟲 oxygen scavenging system 表現的影響 31
第五章 討論 33
參考文獻 41
附錄 1儀器與材料 73
附錄 2 Oligo GEArray Human Common Cytokines Microarray 77
附錄 3 Oxygen scavenging system of T. vaginalis 78
附錄 4 Publications 79
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