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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹智強(Chih-Chiang Chan) | |
| dc.contributor.author | Chen-Yi Wu | en |
| dc.contributor.author | 吳貞儀 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:44:01Z | - |
| dc.date.available | 2021-07-10T21:44:01Z | - |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-07-21 | |
| dc.identifier.citation | 1. Simons, M. and G. Raposo, Exosomes--vesicular carriers for intercellular communication. Curr Opin Cell Biol, 2009. 21(4): p. 575-81.
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Alonso, Biophysical properties of sphingosine, ceramides and other simple sphingolipids. Biochem Soc Trans, 2014. 42(5): p. 1401-8. 28. Chan, J.P., Z. Hu, and D. Sieburth, Recruitment of sphingosine kinase to presynaptic terminals by a conserved muscarinic signaling pathway promotes neurotransmitter release. Genes Dev, 2012. 26(10): p. 1070-85. 29. Ndoye, A. and A.T. Weeraratna, Autophagy- An emerging target for melanoma therapy. F1000Res, 2016. 5. 30. Parzych, K.R. and D.J. Klionsky, An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signal, 2014. 20(3): p. 460-73. 31. Fader, C.M., et al., Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in k562 cells. Traffic, 2008. 9(2): p. 230-50. 32. Dias, M.V., et al., PRNP/prion protein regulates the secretion of exosomes modulating CAV1/caveolin-1-suppressed autophagy. Autophagy, 2016. 12(11): p. 2113-2128. 33. Minakaki, G., et al., Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype. Autophagy, 2018. 14(1): p. 98-119. 34. Xu, J., R. Camfield, and S.M. Gorski, The interplay between exosomes and autophagy - partners in crime. J Cell Sci, 2018. 131(15). 35. Hurwitz, S.N., et al., Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1. J Virol, 2018. 92(5). 36. Hernández-Tiedra, S., et al., Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization. Autophagy, 2016. 12(11): p. 2213-2229. 37. Löfgren, H. and I. Pascher, Molecular arrangements of sphingolipids. The monolayer behaviour of ceramides. Chem Phys Lipids, 1977. 20(4): p. 273-84. 38. Pascher, I., Molecular arrangements in sphingolipids. Conformation and hydrogen bonding of ceramide and their implication on membrane stability and permeability. Biochim Biophys Acta, 1976. 455(2): p. 433-51. 39. Guo, B.B., S.A. Bellingham, and A.F. Hill, The neutral sphingomyelinase pathway regulates packaging of the prion protein into exosomes. J Biol Chem, 2015. 290(6): p. 3455-67. 40. Haraszti, R.A., et al., High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources. J Extracell Vesicles, 2016. 5: p. 32570. 41. Skotland, T., et al., Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology. J Lipid Res, 2019. 60(1): p. 9-18. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77032 | - |
| dc.description.abstract | 外泌體在神經退化性疾病中扮演重要的角色，且近期研究顯示外泌體的釋放可能和膜上的神經醯胺含量有關。Ifc參與神經脂質(sphingolipid)從頭合成路徑中，具有酵素催化功能，可將二羥基神經醯胺(dihydroceramide, dhCer)轉換成神經醯胺(ceramide, Cer)。有實驗發現提高神經醯胺的量可能會增加外泌體的釋放，且先前實驗室研究在果蠅幼蟲眼碟利用背側-眼睛轉錄因子系統(DE-Gal4 system)，以mCD63作為外泌體標定蛋白，發現過表達ifc會使細胞外mCD63訊號增加；反之，當失去ifc時，則會減少細胞外mCD63的訊號，因此顯示ifc可能會調控外泌體的釋放。現今研究發現ILV的形成由不只一種機制調控，且因一般對於外泌體的研究都會使用兩種以上的外泌體標定蛋白，所以我使用另外兩種外泌體標定蛋白：hTSG101和Flo2，來區分不同ILV生合成的專一性，實驗結果同樣發現過表達ifc會使細胞外hTSG101訊號增加，當失去ifc時，則會減少細胞外Flo2的訊號。利用穿透式電子顯微鏡觀察果蠅成蟲眼睛，發現在失去ifc的情況下，單位面積之多囊泡體(multivesicular body, MVB)中的腔內囊泡(intraluminal vesicle, ILV)數量下降，顯示ifc可能透過影響腔內囊泡的形成，調控外泌體的釋放。
有研究發現增加神經醯胺的含量會促使ILV的生成，因此我假設ifc會透過其酵素催化功能，將二羥基神經醯胺(dihydroceramide, dhCer)轉換成神經醯胺(ceramide, Cer)，提高神經醯胺的量，增加腔內囊泡的形成，進而增加外泌體的 釋放，所以我將含有二羥基神經醯胺的巨大單層膜囊泡(giant unilamellar vesicle, GUV)和DEGS1培養，發現會促使腔內囊泡的形成，然而加入fenretinide阻斷DEGS1的酵素活性後，則抑制腔內囊泡的產生，顯示ifc可能透過其酵素催化功能調控外泌體的釋放。 有文獻指出在活體外(in vitro)促進細胞自噬作用可能會抑制外泌體的釋放，因此我探討在活體內(in vivo)的神經細胞中ifc如何調控外泌體釋放及細胞自噬作用，我利用3-甲基腺嘌呤 (3-Methyladenine, 3-MA)和氯喹(chloroquine, CQ)分別抑制細胞自噬作用的前期及中後期，並以mCD63作為外泌體標定蛋白，實驗發現以藥物抑制細胞自噬作用，會增加控制組外泌體的分泌，然而在過表達ifc的情況下，以藥物抑制細胞自噬作用，並沒有更增加外泌體的釋放，顯示抑制細胞自噬作用會使外泌體釋放增加，且過表達ifc與抑制細胞自噬作用對促進外泌體的釋放有相同效果。本研究指出Ifc可能於MVB藉由調控神經醯胺的產生而控制外泌體的生成，其角色類似控制細胞外泌以及細胞自噬的轉換器(switch)。 | zh_TW |
| dc.description.abstract | Ceramide promotes exosome biogenesis, which is crucial for several physiological events including neurodegeneration. Ifc (Infertile crescent) protein converts dihydroceramide to ceramide in the sphingolipid de novo synthesis pathway. We previously utilized the Dorsal-Eye Gal4 (DE-Gal4) system to express both wild-type ifc-mCherry and GFP-mCD63 in the dorsal part of Drosophila eye disc and found an increased in the number of mCD63 puncta in a distant region, suggesting Ifc promotes exosome secretion. In contrast, knocking out of ifc inhibited the secretion of mCD63. To further validate the role Ifc plays in exosome secretion, I used two exosome markers (hTSG101 and Flo2). Overexpressing wild-type ifc-mCherry increased the number of hTSG101 puncta in a distant region, while ifc knockout decreased the secretion of Flo2. Furthermore, in the TEM images of wild-type and ifc-KO Drosophila photoreceptors we found that the ILV density decreased in ifc-KO MVB. I therefore concluded that Ifc controls both the biogenesis and secretion of exosome.
I then investigated the catalytic activity of DEGS1 regulating ILV biogenesis in vitro. GUVs which were generated with dihydroceramide incubated with DEGS1 induce ILVs formation, while adding the DEGS1 inhibitor fenretinide suppressed the formation of ILVs induced by dhCer and DEGS1, indicating that the catalytic activity of DEGS1 was required to induce the formation of ILV in the GUVs. To investigate the interplay between autophagy and exosome, I fed a mixture of normal food and autophagy inhibitor (3-MA or CQ) to Drosophila larvae with the expression of wild-type ifc-mCherry and GFP-mCD63 in the Drosophila eye disc with DE-Gal4. Overexpressing mCherry-CAAX and treating with 3-MA or CQ increased the secretion of mCD63, indicating that inhibition of autophagy may induce exosome secretion. However overexpressing wild-type ifc-mCherry and treating with 3-MA or CQ did not increase the secretion of mCD63, indicating that Ifc may inhibit autophagy and induce exosome secretion. These data indicate that Ifc converts dihydroceramide to induces ILV biogenesis, suggesting its functional role as a switch between exosome secretion and autophagy in neuronal tissue. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:44:01Z (GMT). No. of bitstreams: 1 U0001-2107202010162700.pdf: 2864229 bytes, checksum: 8f995081f5630d218344292470bcd470 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 致謝................................................................................................................................i
摘要...............................................................................................................................ii Abstract ........................................................................................................................iv 目錄..............................................................................................................................vi 第一章 實驗背景.........................................................................................................1 1.1外泌體在神經退化性疾病中所扮演的角色 .....................................................2 1.2外泌體的合成路徑及機制 .................................................................................2 1.3 Ifc和神經脂質的功能........................................................................................4 1.4 Ifc酵素活性調控外泌體釋放............................................................................6 1.5外泌體釋放和細胞自噬作用的關係 .................................................................8 1.6 小結 ....................................................................................................................9 第二章 實驗材料與方法...........................................................................................11 2.1 果蠅株及培養方法 ..........................................................................................12 2.2 果蠅食物培養基製備 ......................................................................................13 2.3免疫螢光染色及共軛焦顯微鏡的使用 ...........................................................13 2.4免疫螢光染色抗體清單 ...................................................................................14 2.5果蠅餵食藥劑實驗製備 ...................................................................................14 2.6 胞外囊泡(extracellular vesicles, EVs)的製備...............................................15 2.7巨大單層膜囊泡 (giant unilamellar vesicle, GUV)的合成 ............................15 第三章 實驗結果.......................................................................................................16 3.1過表達ifc(WT)-mCherry使細胞外hTSG101 puncta數量增加 ....................17 3.2失去ifc使細胞外Flo2訊號減少....................................................................17 3.3奈米顆粒追蹤分析粒徑大小約為100 nm......................................................18 3.4失去ifc使單位面積多囊體中的腔內囊泡數量下降 .....................................18 3.5含有神經醯胺的GUV其含有ILVs的比例較高 ..........................................19 3.6 DEGS1的催化使帶有二羥基神經醯胺的GUV產生ILVs..........................19 3.7 DEGS1無法催化不含二羥基神經醯胺的GUV產生ILVs..........................20 3.8 Fenretinide抑制DEGS1使帶有二羥基神經醯胺的GUV無法產生ILV s 20 3.9失去ifc可能促進細胞自噬作用 .....................................................................21 3.10餵食3-MA抑制細胞自噬作用起始會增加細胞外mCD63 puncta的數量21 3.11餵食CQ抑制細胞自噬作用後期會增加細胞外mCD63 puncta的數量 ...22 第四章 實驗討論.....................................................................................................24 4.1 Ifc可能和ESCRT蛋白共同調控外泌體的釋放 ...........................................25 4.2脂質成分對於外泌體的影響 ...........................................................................25 4.3過表達ifc可能會抑制細胞自噬作用 .............................................................26 4.4決定多囊泡體命運的關鍵步驟 .......................................................................26 4.5 GUV脂質組成是否影響腔內囊泡的形成 .....................................................27 第五章 未來實驗方向.............................................................................................28 5.1探討Ifc/DEGS1和ESCRT蛋白如何協同調控外泌體的釋放 ....................29 5.2探討DEGS1是否會調控細胞自噬作用 ........................................................29 5.3分析神經細胞多囊泡體脂質的組成 ...............................................................30 第六章 實驗圖表.....................................................................................................31 Fig. 1過表達ifc(WT)-mCherry使細胞外hTSG101-HA puncta數量增加 ........32 Fig. 2失去ifc使細胞外Flo2訊號減少 ...............................................................33 Fig. 3奈米顆粒追蹤分析(Nanoparticle tracking analysis, NTA)外泌體粒徑大小 .................................................................................................................................34 Fig. 4失去ifc使單位面積多囊體中的腔內囊泡數量減少 ................................35 Fig. 5含有神經醯胺的GUV其含ILVs的比例較高 .............................................36 Fig. 6 DEGS1蛋白的酵素催化功能對於腔內囊泡的生合成是必須的 .............37 Fig. 7失去ifc使mCD63和Atg8a訊號的共位增加 ..........................................38 Fig. 8抑制細胞自噬作用會增加細胞外mCD63 puncta的數量 ........................39 Supplementary Fig. 1餵食3-MA會抑制細胞自噬作用......................................41 第七章 參考文獻.......................................................................................................4 | |
| dc.language.iso | zh-TW | |
| dc.subject | 二羥基神經醯胺 | zh_TW |
| dc.subject | 外泌體 | zh_TW |
| dc.subject | 自噬作用 | zh_TW |
| dc.subject | 神經醯胺 | zh_TW |
| dc.subject | autophagy | en |
| dc.subject | exosome | en |
| dc.subject | Infertile crescent | en |
| dc.subject | dihydroceramide | en |
| dc.subject | ceramide | en |
| dc.title | infertile crescent (ifc) 於果蠅眼碟調控外泌體合成、釋放及細胞自噬之研究 | zh_TW |
| dc.title | Investigating the role of infertile crescent (Ifc) in exosome biogenesis and autophagy in Drosophila eye disc | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉雅雯(Ya-Wen Liu),李芳仁(Fang-Jen Lee),沈湯龍(Tang-Long SHEN),黃舒宜(Shu-Yi Huang) | |
| dc.subject.keyword | 外泌體,自噬作用,神經醯胺,二羥基神經醯胺, | zh_TW |
| dc.subject.keyword | Infertile crescent,exosome,autophagy,ceramide,dihydroceramide, | en |
| dc.relation.page | 47 | |
| dc.identifier.doi | 10.6342/NTU202001680 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-07-21 | |
| 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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