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研究生:黃于恬
研究生(外文):Yu-Tien Huang
論文名稱:探討第八型岩藻醣轉移酶藉活化表皮生長因子受體而調控角質細胞增生
論文名稱(外文):Fucosyltransferase 8 (FUT8) Mediates Epidermal Growth Factor Receptor (EGFR) Activation to Regulate Keratinocyte Proliferation
指導教授:李永凌李永凌引用關係
口試日期:2017-07-25
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:流行病學與預防醫學研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:43
中文關鍵詞:乾蘚第八型岩藻醣轉移酶醣基化表皮細胞生長因子受體細胞增生角質細胞
外文關鍵詞:PsoriasisFucosyltransferase 8GlycosylationEpidermis growth factor receptorProliferationKeratinocyte
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研究目的及方法:
乾蘚是一個常見的慢性遺傳性皮膚疾病,其主要特徵為表皮增生分化出現不正常。表皮細胞生長因子受體為表皮細胞膜上與細胞增生高度相關的蛋白,此受體及其主要配體已經被發現在乾蘚病灶處大量表達。在哺乳動物體內,第八型岩藻醣轉移酶是唯一可以對蛋白質進行核心岩藻醣基化的酵素。在過去的實驗當中,我們證實在人類角質細胞中的第八型岩藻醣轉移酶可以促進細胞的增生。表皮細胞生長因子受體為一醣基化蛋白,並已證實在多種癌症中受到第八型岩藻醣轉移酶調控。因此本研究旨在討論人類角質細胞中經由第八型岩藻醣轉移酶調控的表皮細胞生長因子受體活化反應。
首先經由臨床資料與檢體分析乾蘚病灶與非病灶中第八型岩藻醣轉移酶的表現量,並計算其與臨床嚴重程度的相關性。其次經由醣蛋白體學技術找出第八型岩藻醣轉移酶在其變異細胞株中的關鍵醣蛋白。並經由免疫染色等方式呈現第八型岩藻醣轉移酶變異細胞株中與正常角質細胞相異的表皮細胞生長因子受體其本身與下游磷酸化訊息傳遞鍊,二聚體構成,配體結合力以及表面受體內部化等等活化反應。
結果與結論:
免疫染色結果顯示乾蘚病灶相較於非病灶中有較高的第八型岩藻醣轉移酶表現量,且其表現量比值與臨床嚴重程度成正向關係。由醣蛋白體學技術找出第八型岩藻醣轉移酶變異細胞株中,表皮細胞生長因子受體的核心岩藻醣受到調控。在生長因子配體刺激下,表皮細胞生長因子受體其本身與下游磷酸化訊息傳遞鍊,二聚體構成,配體結合力以及表面受體內部化等等活化反應在第八型岩藻醣轉移酶弱化的細胞株中都呈現較正常細胞株低下的反應,而這些影響則間接造成角質細胞增生異常。
Background:
Psoriasis is a common, heritable, and chronic inflammatory skin disease. Epidermal hyperplasia is directly caused by keratinocyte hyperproliferation. Epidermis growth factor receptor (EGFR), a transmembrane receptor highly related with cell proliferation, and its ligands were reported to be up-regulated in psoriasis lesion skin. Fucosyltransferase 8 (FUT8) is the only enzyme to catalyze 1,6-fucosylation in mammal and has been observed to regulated cell proliferation. Since EGFR is a core fucosylated protein, we aim to investigate the regulatory role of FUT8 on EGFR activation.
Method:
FUT8 expression in psoriasis was examined with immunofluorescence. In vitro studies were conducted under lentiviral transduction of FUT8 overexpress and knockdown on normal human keratinocyte cell line (HaCaT). Core fucosylated glycoproteins were isolated by LC MS/MS with SILAC labeling. Immunoprecipitation and immunoblotting were used to investigate EGFR core fucosylation, phosphorylation and downstream signaling. BS3 cross linker was used to observe EGFR dimerization. Alkaline phosphatase report system was used to investigate EGF binding affinity. Alexa-488 conjugated EGF was used to observe EGFR internalization.
Results:
FUT8 was up-regulated in psoriasis lesion skin. Knockdown of FUT8 suppresses EGFR core fucosylation, EGFR phosphorylation, MAPK signaling, EGFR dimerization, EGF binding affinity and EGFR internalization in human keratinocyte cell line (HaCaT).
1. INTRODUCTION 6
1.1 INTRODUCTION TO PSORIASIS 6
1.2 FUT8 IN CELL PROLIFERATION 7
1.3 EGFR IN CELL PROLIFERATION AND PSORIASIS 8
1.4 EGFR ACTIVATION IN FUT8 MUTANT CELL 9
2. SPECIFIC AIM 10
3. MATERIAL AND METHOD 11
3.1 MATERIALS 11
3.2 STUDY POPULATION AND CLINICAL PSORIASIS STATUS EVALUATION 11
3.3 MICROSCOPY, IMMUNOFLUORESCENCE (IF) AND IMAGE ANALYSIS 12
3.4 STATISTICAL ANALYSIS 13
3.5 CELLS AND CELL CULTURE 13
3.6 IMMUNOPRECIPITATION AND IMMUNOBLOT 13
3.7 EGF-EGFR COMPLEX INTERNALIZATION 14
3.8 EGFR DIMERIZATION ASSAY 15
3.9 EGF LIGAND BINDING ASSAY 15
4. RESULT 17
4.1 HIGHER FUT8 EXPRESSION LEVEL IN PSORIASIS LESION AND MORE SEVERE PSORIASIS 17
4.2 EGFR IS A KEY RECEPTOR REGULATED BY FUT8 IN HUMAN KERATINOCYTE 18
4.3 KNOCKDOWN OF FUT8 SUPPRESSES LIGAND INDUCED ERK PATHWAY IN HACAT 21
4.4 KNOCKDOWN OF FUT8 SUPPRESSES LIGAND INDUCED EGFR DIMERIZATION IN HACAT 22
4.5 KNOCKDOWN OF FUT8 SUPPRESSES EGF BINDING AFFINITY IN HACAT 23
4.6 KNOCKDOWN OF FUT8 DELAYS EGF INDUCED EGFR INTERNALIZATION IN HACAT 24
5. DISCUSSION 26
5.1 THE ROLE OF FUT8 IN EGFR REGULATION 26
5.2 EGFR REGULATION OF FUT8 IN PSORIASIS ANIMAL MODEL 27
5.3 FUTURE TRANSLATIONAL PERSPECTIVE 27
6. REFERENCE 28
7.
FIGURE 1. FUT8 WAS UP-REGULATED IN PSORIASIS LESION AND HIGHER LESION/NON LESION RATIO OF FUT8 IN MORE SEVERE PSORIASIS 31
FIGURE 2. FUT8 OVEREXPRESS AND KNOCKDOWN WITH LENTIVIRAL TRANSDUCTION. HUMAN KERATINOCYTE PROLIFERATION WAS REGULATED BY FUT8 32
FIGURE 3. DECREASED LCA STAINED SURFACE CORE FUCOSE IN FUT8 KNOCKDOWN HACAT. 33
FIGURE 4. PHOSPHORYLATED EGFR AND SIX OTHERS RECEPTOR TYROSINE KINASES WERE UP-REGULATED IN FUT8 OVEREXPRESSED HACAT CELL. 34
FIGURE 5. CORE FUCOSYLATED GLYCOPROTEINS ISOLATION AND THE CONNECTIVITY MAP OF EGFR AND 38 DIFFERENTIALLY EXPRESSED PROLIFERATION RELATED GENES. 35
FIGURE 6. KNOCKDOWN OF FUT8 SUPPRESSES EGFR CORE FUCOSYLATION AND PHOSPHORYLATION 36
FIGURE 7. KNOCKDOWN OF FUT8 SUPPRESSES LIGAND INDUCED ERK PATHWAY. 37
FIGURE 8. ELEVATED AKT AND ERK PHOSPHORYLATION IN FUT8 OVEREXPRESS HACAT WERE SUPPRESSED BY EGFR SPECIFIC KINASE INHIBITOR, IRESSA 38
FIGURE 9. KNOCKDOWN OF FUT8 SUPPRESSES LIGAND INDUCED EGFR DIMERIZATION 39
FIGURE 10. KNOCKDOWN OF FUT8 SUPPRESSES EGF BINDING AFFINITY IN HACAT CELL 40
FIGURE 11. EGF BINDING LEVEL BUT NOT SURFACE EXPRESSION LEVEL OF EGFR WAS SLIGHTLY AFFECTED BY KNOCKDOWN OF FUT8 IN HACAT CELL 41
FIGURE 12. KNOCKDOWN OF FUT8 DELAYS EGF INDUCED EGFR TRANSLOCATION TO THE NUCLEUS IN HACAT CELL. 42
FIGURE 13. KNOCKDOWN OF FUT8 SUPPRESSES MAPK SIGNALING, EGFR DIMERIZATION, EGF BINDING AFFINITY AND EGFR INTERNALIZATION IN HUMAN KERATINOCYTE 43
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