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系統識別號 U0026-3006201019185000
論文名稱(中文) Fas Ligand活化Akt與NF-kB並調控細胞激素的表現
論文名稱(英文) Fas ligand signal activates Akt and NF-kB and mediates cytokines expression
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) 廖宏明
研究生(英文) Hong-Ming Liao
學號 s4696111
學位類別 碩士
語文別 英文
論文頁數 55頁
口試委員 指導教授-楊倍昌
口試委員-張晃猷
口試委員-沈延盛
中文關鍵字 FasL  MCF-7  MCP-1  IL-8 
英文關鍵字 FasL  MCF-7  MCP-1  IL-8 
學科別分類
中文摘要 FasL是一個廣為人知的穿膜蛋白,它最著名的功能是與Fas結合進而引發細胞凋亡。之前的研究發現,FasL在皮下腫瘤內的高量表現,會伴隨著大量的嗜中性球浸潤。而我們發現,使用FasL ribozyme降低FasL在B16F10細胞內的表現量,也會使MIP-2和TGF-beta的表現量降低。而在人類乳癌細胞MCF-7內大量表現FasL,則可發現IL-8和MCP-1的表現量上升,並且使用泛caspase抑制劑並不會改變其影響。使用截去細胞質端CKI結合區域和截去CKI結合區域與proline rich domain的FasL,我們發現所有FasL皆可以使MCP-1和IL-8的表現量上升,但是帶有proline rich domain的FasL上升的量較多。進一步地,我們發現在送入帶有proline rich domain的FasL之MCF-7 細胞中,Akt有被活化的現象,其下游的GSK3同時受到抑制,並且RANTES的表現也同時受到抑制。以免疫沉澱法並沒有發現FasL與PI3K有結合作用的現象。觀察NF-kB的活化情形,可以發現不論在FasL或是截去細胞質端的FasL,都有IkB的磷酸化並被降解的現象。使用PI3K抑制劑LY294002與NF-kB抑制劑SN50,可以使IL-8和MCP-1的表現都被抑制,但是只有使用LY294002,才會使用RANTES被抑制的結果回復。以上結果的結論是,FasL可以傳遞兩種訊息,一種是透過proline rich domain活化NF-kB和Akt,隨後抑制GSK3的活性並使RANTES的表現量下降,另一種是透過Akt-independent的方式活化NF-kB,使IL-8和MCP-1表現量增加。
英文摘要 FasL is a well known death-inducing transmembrane protein which binds to Fas and induces cell apoptosis. Previous studies on subcutaneous tumor show that overexpression of FasL contributes to abundant neutrophils infiltration. We found that using FasL ribozyme to reduce the expression level of FasL in B16F10 cells would decrease the expresson of MIP-2 and TGF-beta. Overexpression of FasL in human breast cancer MCF-7 led to an increase in IL-8 and MCP-1 expression, and this induction was not altered by using pan-caspase inhibitor. Using truncated FasL deleting cytoplasmic CKI binding motif or both CKI binding motif and proline rich domain, we found that FasL increased the expression of IL-8 and MCP-1, and deleting proline rich domain resulted in a decrease in the expression of IL-8 and MCP-1. In addition, we found that ectopic expression of FasL activated Akt and elevated GSK3 phosphorylation accompanied by downregulation of RANTES. No interation of FasL and PI3K was found by immunoprecipitation assay. In addition, we found that IkB was phosphorylated and degraded in MCF-7 expressing FasL or truncated FasL. PI3K inhibitor LY294002 and NF-kB inhibitor SN50 inhibited the expression of IL-8 and MCP-1. Treatment of LY294002 reverted the RANTES expression. In conclusion, FasL transduced two signals, one signal required proline rich domain and activated NF-kB and Akt, which subsequently inhibited GSK3 activity and RANTES expression, another resulted in Akt-independent NF-kB activation and the expressions of IL-8 and MCP-1.
論文目次 摘要 I
Abstract III
誌謝 V
Contents VI
Figure Index VIII
Abbreviations IX
Introduction 1
Materials & Methods 6
Materials 7
Media & Buffers 11
Methods 17
Plasmids extration 17
Cell culture 18
Transfection 19
Western-Blot analysis 20
Reverse transcriptase-polymerase chain reaction 22
Immunoprecipitation 26

Results 29
Fas ligand involved in cytokines production
in B16F10 29
Ectopic expression of FasL induced
caspase-independent cytokines expression 29
Expression of ectopic FasL or Fas
in MCF-7 cells 30
Trucated FasL induced IL-8 and MCP-1 expresion 31
Proline rich domain of FasL activated
Akt phosphorylation 31
FasL induced IkB phosphorylation
and degradation 31
Proline rich domain of FasL inhibited
GSK3 activity and RANTES expression 32
FasL-PI3K interaction was not detected by immunoprecipitation in MCF-7 transfectants 33
The alteration of PI3K and NF-kB acitivity 33
Discussion 34
References 36
Figures 40
自述 55
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