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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 余佳慧(Chia-Hui Yu) | |
| dc.contributor.author | Chi-Yun Chen | en |
| dc.contributor.author | 陳霽雲 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:52:06Z | - |
| dc.date.available | 2013-09-09 | |
| dc.date.copyright | 2010-09-09 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-07-31 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46040 | - |
| dc.description.abstract | 背景：脂多醣 (lipopolysacchraide, LPS)為格蘭氏陰性菌細胞外膜的組成成份之一。當免疫細胞(如：單核球、巨噬細胞)透過其細胞表面的 LPS受器複合體 (receptor complex)—CD14/TLR4/MD2辨認出 LPS時，會誘導促發炎 (proinflammatory)反應的發生。然而， CD14與 TLR4在人類腸道上皮細胞截然不同的表現型態似乎正顯示著一項腸腔之所以能夠容忍腸道共生菌叢存在的機制。本實驗室先前的研究已發現，自腔面 (luminal side)給予 LPS刺激會導致人類腸道上皮細胞株 Caco-2的凋亡與緊密連結 (tight junction)的斷裂1-2；但是這些反應與 TLR4訊息傳導途徑無關(未發表之實驗結果)。近期於免疫細胞的研究證據指出，LPS和其細胞膜表面的 CD14分子的結合後，將啟動一串脂質第二傳訊分子的傳導途徑。磷脂膽鹼磷脂酶 C (phosphatidylcholine-specific phospholipase C, PC-PLC)的活化可造成甘油二酯 (diacylglycerol, DAG)的生成，進一步催化神經磷脂酶 (sphingomyelinase, SMase)的活化，使其將神經磷脂 (sphingomyelin)轉化成神經醯胺 (ceramide)。 Ceramide的產生將活化蛋白質激酶 Cζ (protein kinase Cζ, PKCζ)，並導致 TLR4被趨化至脂筏上，以和 CD14形成受器複合體。已知 Ceramide和 PKCζ在許多種類的上皮細胞中均與促凋亡 (pro-apoptotic)反應有關。然而， CD14媒介的訊息傳遞是否參與 LPS所誘導的腸道上皮細胞凋亡反應目前尚未可知。材料與方法：採用長至全滿之 Caco-2細胞株 (C2BBe clone)，自其腔面給予數個不同濃度與不同時間點的 LPS刺激；而某些組別在 LPS刺激前先以藥物性抑制劑，包括 D609 (PC-PLC抑制劑)、鹽酸丙咪嗪 (imipramine，SMase抑制劑)、 Gö6983(廣效性的 PKC抑制劑)與抑制性的 PKCζ偽受質 (inhibitory PKCζ pseudosubstrate)預處理細胞。細胞凋亡的現象透過缺口末端標記技術來觀察，並以細胞凋亡酶連免疫分析法來量化之。 PKCζ的活化狀態以免疫螢光共軛焦顯微鏡技術與西方轉漬法來分析。並使用極致液相層析—連續質譜儀系統 (Ultra-performance liquid chromatography–tandem mass spectrometry, UPLC—MS/MS)來偵測ceramide的產生。 CD14與 TLR4的蛋白質和 mRNA表現量則使用免疫螢光染色法和反轉錄酶-聚合酶鏈反應來檢驗之。實驗結果：無論在 LPS刺激前或刺激後， Caco-2細胞頂腔面 (apical)的 CD14蛋白質染色結果表現穩定，而 TLR4蛋白質則始終付之闕如。自腔面給予 Caco-2細胞 LPS的刺激可誘導其發生凋亡反應，且凋亡的程度有時間與劑量依賴性(time and dose-dependent)。藥物性抑制劑 D609、 imipramine、 Gö6983和 inhibitory PKCζ pseudosubstrate的預處理可有效減少受 LPS刺激增加的細胞凋亡程度。 Ceramide (C16)的產量在 LPS刺激後亦有顯著的增加。此外， LPS可引發胞內 PKCζ蛋白質的磷酸化，並促進其移位 (translocation)至細胞表面，而與緊密連結有共位 (colocalization)的現象；更甚者，這些現象也會被D609與 imipramine的預處理所抑制。結論： LPS誘導的腸道上皮細胞凋亡與 CD14媒介的訊息傳導途徑，如 ceramide與 PKCζ有關。 | zh_TW |
| dc.description.abstract | Background: Lipopolysaccharide (LPS) is a component of the outer membrane of Gram (-) bacteria. Proinflammatory responses are mounted by immune cells (e.g. monocyte/macrophage) following LPS recognition by its receptor complex (CD14/TLR4/MD2) on cell surface. However, distinct expression patterns of CD14 and TLR4 were characterized on human intestinal epithelial cells, which were suggested as a mechanism to tolerate against commensal bacteria in the gut lumen. Previous studies from our laboratory have demonstrated that luminal LPS challenge induced epithelial cell apoptosis and tight junctional breakage in human intestinal Caco-2 cells1-2, of which the mechanism was independent of TLR4 signaling (unpublished data). Recent evidence on monocytic cell lineages showed that after LPS binding to membranous CD14, a cascade of lipid secondary messenger pathway occurred. Activation of phosphatidylcholine-specific phospholipase C (PC-PLC) caused the generation of diacylglycerol (DAG) which then catalyzed sphingomyelinase to convert sphingolipid to ceramide. Formation of ceramide activated PKCζ leading to recruitment of TLR4 to lipid rafts to form a complex with CD14. Both ceramide and PKCζ were implicated in pro-apoptotic pathways in various epithelial cell types. It remains unknown whether CD14-mediated signals are involved in the mechanism of LPS-induced apoptosis in intestinal epithelial cells. Materials and methods: Caco-2 cells (C2BBe clone) were grown to confluency and apically challenged with LPS at various concentration and time points. In some groups, cells were pretreated with pharmacological inhibitors, such as D609 (a PC-PLC inhibitor), imipramine (a sphingomyelinase inhibitor), Gö6983 (a broad spectrum PKC inhibitor), or inhibitory PKCζ pseudosubstrate prior to LPS challenge. Cell apoptosis was assessed by TUNEL staining and quantified using a Cell Death ELISA kit. The activation status of PKCζ was examined by western blotting and immunofluorescent confocal microscopy. Production of ceramide was detected by Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC—MS/MS). The expression levels of CD14 and TLR4 proteins and mRNA were examined by immunofluorescent staining and RT-PCR. Results: Apical expression of CD14 and absence of TLR4 staining was confirmed on Caco-2 cells before and after LPS challenge. Exposure to luminal LPS increased the level of apoptosis in Caco-2 cells in a time- and dose-dependent manner. Pretreatment with D609, imipramine, Gö6983, and inhibitory PKCζ pseudosubstrate decreased the cell apoptotic levels induced by LPS. The level of ceramide (C16) was significantly elevated after LPS challenge. Moreover, LPS triggered increased phosphorylation and membrane translocation of cytosolic PKCζ to colocalize with tight junctions. The activation of PKCζ was blocked by pretreatment with D609 and imipramine. Conclusion: LPS-induced intestinal epithelial apoptosis is dependent on CD14-mediated signaling pathways such as ceramide and PKCζ. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:52:06Z (GMT). No. of bitstreams: 1 ntu-99-R96441004-1.pdf: 5796646 bytes, checksum: c555d20731444c8f20a53ffe03e5895f (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 中文摘要 IV Abstract VI 名詞中英文縮寫對照表 VIII 目錄 XI 圖表目錄 XIV 一、前言 1 1、腸道上皮細胞概述 1 1.1 物理性屏障 1 1.2 化學性屏障 2 1.3免疫性屏障 2 2、腸道上皮細胞之新生與凋亡 3 2.1 腸道腺窩-絨毛軸 (crypt-villus axis) 3 2.2 細胞凋亡概述 4 2.3上皮細胞過量凋亡造成屏障功能喪失 6 2.4 上皮細胞凋亡不足為癌化原因之一 6 3、細菌對腸道上皮細胞替換率 (turnover rate)及細胞凋亡量之影響 7 3.1 正常腸腔共生菌叢 7 3.2 病原菌的感染 8 4、脂多醣受器複合體—CD14/TLR4/MD-2及其訊息傳導途徑 9 4.2 LPS受器複合體在免疫細胞上的表現 9 4.3 CD14 和TLR4在上皮細胞的表現 10 4.3.1 正常人和病人腸道檢體之上皮細胞 CD14 和 TLR4 表現 10 4.3.2 人類腸道上皮癌細胞株中的 CD14和 TLR4表現量和位置 11 5、 LPS訊息傳導途徑 12 5.1. TLR4下游的訊息傳導 12 5.2 CD14下游的訊息傳導 13 5.2.1 PLC 14 5.2.2 Ceramide 14 5.2.3 PKC 15 5.2.4 PKCζ 17 6. 腔面LPS刺激引發腸道上皮細胞之凋亡增加 18 6. 1 TLR4下游訊息傳導途徑與細胞凋亡之關係 18 6. 2 CD14下游訊息傳導途徑與細胞凋亡之關係 19 7、研究緣由及目的 20 二、材料與方法： 21 1、細胞培養 21 2、大腸桿菌脂多醣 (Lipopolysaccharide, LPS) 21 3、抑制劑 22 3.1 蛋白質激酶 C (Protein kinase C)抑制劑： 22 3.1.1 Gö6983 22 3.1.2 抑制性的蛋白質激酶 Cζ偽受質 (Inhibitory PKCζ pseudosubstrate (PKCζ-ps), myristoylated) 22 3.2 D609 23 3.3 Imipramine hydrochloride 23 4、核醣核酸的萃取與反轉錄酶—聚合酶鏈反應 (RNA Extraction & Reverse Transcription Polymerase Chain Reaction, RT-PCR) 24 4.1 核醣核酸的萃取 (RNA Extraction) 24 4.1.1 均質 (Homogenization) 24 4.1.2 相位分離 (Phase Seperation) 24 4.1.3 RNA沉澱反應 (RNA Precipitation) 24 4.1.4 清洗 RNA (RNA Wash) 24 4.1.5 回溶 RNA (Redissolving RNA) 25 4.2 反轉錄反應 (Reverse Transcription, RT) 25 4.3 聚合酶鏈反應 (Polymerase Chain Reaction, PCR) 25 4.4 膠體製備 (Gel Preparation) 26 4.5 電泳 (Electrophoresis) 26 5、缺口末端標記技術 (TdT-mediated dUTP-biotin Nick End Labeling, TUNEL assay) 27 6、細胞凋亡酶連免疫分析法 (Cell Death Detection ELISA) 28 7、蛋白質萃取暨西方轉漬分析法 (Protein Extraction and Western Blotting)： 29 7.1 樣本製備：細胞質蛋白質之萃取 (Extraction of Cytoplasmic Proteins)： 29 7.2 西方轉漬分析法 (Western Blotting) 29 7.2.1 膠體製備 (Gel Preparation) 29 7.2.2 電泳 (Gel Electrophoresis) 30 7.2.3 轉漬 (Transferring) 30 7.2.4 封鎖 (Blocking)、一級抗體與二級抗體免疫結合 31 7.2.5 呈色、影像擷取、半定量 31 8、免疫螢光染色 (Immunofluorescent Staining) 32 9、內生性神經醯胺分析 (Endogenous Ceramide Analysis)： 33 9.1 脂質的萃取 (Lipid Extraction)： 33 9.2 極致液相層析—連續質譜儀系統 (Ultra Performance Liquid Chromatography [UPLC]—Tandem Mass Spectrometry System)： 34 9.2.1 實驗原理 34 9.2.2 儀器 35 9.2.3 待測脂質標準品之製備： 35 9.2.4操作條件 36 10、統計分析方法 (Statistics) 36 三、結果 37 1、 人類大腸上皮Caco-2細胞株有 CD14，而無 TLR4的表現 37 2、 自腔面給予 LPS刺激會造成 Caco-2細胞凋亡，並呈現劑量與時間依賴性 37 3、預處理 PC-PLC、 SMase、 PKCζ抑制劑可降低 LPS刺激造成的 Caco-2細胞凋亡量 38 4、 LPS的刺激可誘導 Caco-2細胞 C16 ceramide的增加 38 4.1 Caco-2細胞中可偵測到生理態 ceramide C16的表現 38 4.2 Caco-2細胞的 C16 ceramide隨 LPS的刺激而增加 39 5、 自腔面給予 LPS刺激造成 Caco-2細胞的 PKCζ之活化 39 四、討論 41 五、附圖與附表 46 六、參考文獻 66 圖表目錄 Table. 1 人類腸道上皮細胞檢體的 CD14與 TLR4表現。 46 Table. 2 各種碳鏈長度的 ceramide (C6、 C14、 C16、C18、 C20和 C24)的各項 UPLC-MS/MS參數。 47 Fig. 1 腸道上皮細胞的縱向剖面圖與緊密連結 (tight junction)的結構。 48 Fig. 2免疫細胞(單核球/巨噬細胞)的TLR4下游訊息傳導途徑。 49 Fig. 3 CD14下游的訊息傳導。 50 Fig. 4 Ceramide的分子結構模型與生成途徑。 51 Fig. 5 PKC的分子結構。 52 Fig. 6 PKCζ的分子結構與 PKCζ的活化過程。 53 Fig. 7 Ceramide的分子結構。 54 Fig. 8 Caco-2細胞的 CD14與 TLR4蛋白與 mRNA的表現量。 55 Fig. 9 腸腔面 LPS刺激可造成 Caco-2細胞的 DNA片段化，且此現象呈現劑量與時間依賴性。 56 Fig. 10 腸腔面 LPS的刺激可造成 Caco-2細胞凋亡。 57 Fig. 11 腸腔面 LPS刺激造成 Caco-2細胞凋亡增加的現象可被 D609、imipramine-HCl、 Gö6983與 PKCζ-ps所阻斷。 58 Fig. 12 各式 ceramide標準品的色層分析圖 (chromatogram)。 59 Fig. 13 Caco-2細胞的各式 ceramide色層分析圖。 60 Fig. 14 腸腔面 LPS刺激可引起 Caco-2細胞中的C16 ceramide含量增加。 61 Fig. 15 腸腔面 LPS刺激可引起 Caco-2細胞內磷酸化的 PKCζ (p-PKCζ)含量的增加。 62 Fig. 16 腸腔面 LPS刺激可增加Caco-2細胞的 p-PKCζ 細胞膜交接處表現。 63 Fig. 17 腸腔面 LPS刺激可引起 p-PKCζ分布到細胞膜附近，且此現象可被 D609與 imipramine-HCl所抑制。 64 Fig. 18 腸腔面 LPS刺激可引起 p-PKCζ聚集至細胞膜附近，並與 tight junction產生共位現象。 65 | |
| dc.language.iso | zh-TW | |
| dc.title | 細菌脂多醣經由 CD14誘導腸道上皮細胞凋亡之訊息傳導路徑的探討 | zh_TW |
| dc.title | Signaling Pathways of LPS/CD14-induced Intestinal Epithelial Cell Apoptosis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王錦堂(Jin-Town Wang),繆希椿(Shi-Chuen Miaw),徐立中(Li-Chung Hsu),游偉絢(Wei-Hsuan Yu) | |
| dc.subject.keyword | 腸道上皮細胞,細菌脂多醣,CD14,Toll樣受器4,細胞凋亡,蛋白質激酶,Cζ,神經醯胺, | zh_TW |
| dc.subject.keyword | intestinal epithelial cell(IEC),lipopolysaccharide(LPS),CD14,TLR4,apoptosis,protein kinase Cζ (PKCζ),ceramide, | en |
| dc.relation.page | 90 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-02 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生理學研究所 | zh_TW |
| 顯示於系所單位： | 生理學科所 | |
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