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研究生:李思錦
研究生(外文):Szu Chin, Li
論文名稱:Interleukin- 6透過STAT3機制以啟動頭頸癌鱗狀上皮細胞株Haptoglobin 蛋白的表現
論文名稱(外文):Interleukin-6 Induced STAT3 Signal In HNSCC Cells Promotes Haptoglobin α Subunit Expression
指導教授:黃憲斌 教授
指導教授(外文):Hsien-Bin, Huang
口試委員:李沁廖慧芬
口試委員(外文):Chin, LiHui-Fen, Liao
口試日期:2012-06-15
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:71
外文關鍵詞:HaptoglobinInterleukin-6STAT3
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Haptoglobin (Hp) 是一種急性期蛋白,由肝臟分泌,其濃度在發炎或腫瘤形成時會急速上升。已有相關研究發現血清中Hp的表現型與癌症的發生有其關聯性。過去研究證實,在肝癌細胞中,Hp主要是受到IL-6活化,通過STAT3調控。在人類其構成Hp分子的肽鏈有α和β兩種,Hp多型態主要來自α鏈的遺傳變異,而α鏈有α1和α2兩種,分別產生Hp1和Hp2。口腔癌是常見的癌症,台灣的發生率尤以中南部地區特別高。我們先前的研究結果顯示,在口腔癌病人的血液中之Hp,其 chains的表現量明顯不同,與正常人比較Hp 2上升但Hp 1卻下降。Hp 2-2 genotype的口腔癌病人,其有較高的局部復發機率。我們使用不同基因型的頭頸癌鱗狀上皮細胞株 Hp1-1 以及Hp 2-2,經由IL-6 刺激後,透過STAT3機制調控,獲得個別的Hp 1, 2的表現量。我們發現IL-6刺激下,磷酸化的STAT3增加,接著Hp 增加。而抑制STAT3後,磷酸化的STAT3下降,Hp 也減少。希望能進一步探討Hp與頭頸癌致癌的關連機制與當作可能的生物標的。
Haptoglobin (Hp), a serum α2-sialoglycoprotein, is an acute phase protein secreted by the liver, and its concentration increases rapidly in the status of infection, inflammation, and tumor formation.
The Hp molecule is composed of two subunits, α and β. Two alleles of the α subunit, α1 and α2, constitutes the polymorphism of Hp and
provides distinct biological functions. Our previous research already revealed that the expression level of Hp α alleles was altered in the serum of the head and neck cancer patients, with decreased expression of Hp α1 and increased expression of Hp α2, respectively.
Moreover, the patients carrying the Hp α2 allele have relatively higher probability of recurrence or metastasis.
Thus far, how Hp variants contribute to cancer progression has not been clarified. Therefore, in this study, HNSCC cell lines with distinct Hp alleles were investigated to illustrate the responses of the Hp alleles to immunological signaling. Our result demonstrates that expression of Hp could be activated via Interleukin-6 by activation of transcription factor STAT3.
In the future, we intend to further clarify the signal transduction pathway involved in the Hp-induced carcinogenesis in head and neck cancer and to establish Hp as an effective predictor for cancer progression.


List of Content
摘要………………………………………………………………….… i Abstract………………………………………………………………....ii
List of Content………………………………………………………….iv
List of Figures ………………………………………………………. vi
1. Introduction………………………………………………………… 1
1.1 Introduction of squamous cell carcinoma of the head and neck….1
1.2 The relationship between haptoglobin, interleukin-6 and HNSCC………………………………………………………….. 2
1.3 The hypothesis of the mechanism of the interleukin-6 induces Hp
gene expression via the transcription factor……………………..5
2. Objectives of Study…………………………………………………7
3. Materials and Methods……………………………………………. 8
3.1 Cells and cell lines……………………………………………… 8
3.2 The determination of Haptoglobin polymorphism…………… 8
3.3 Quantitative RT-PCR procedure for Hp α1 and β2 subunit mRNA level …………………………………………………………….10
3.4 Subcellular fractionation………………………………………. 13
3.5 Quantitation of protein………………………………………….14
3.6 Western Blot Analysis………………………………………….14
3.7 STAT 3 inhibitor………………………………………………. 16
3.8 Statistical analysis………………………………………………17
4. Results……………………………………………………………. 18
4.1 The Haptoglobin (Hp) polymorphism in HepG2, FaDu and SCC4
cell lines based on polymerase chain reaction (PCR)………….18
4.2 Effect of IL-6 induced on Hp production by HepG2, FaDu and
SCC4 cell lines………………………………………………...19
4.3 Evaluation of the expression of Hp regulated by IL-6- cytokines
involving STAT3 signal stimulation in HNSCC Cells………...20
4.4 WP1066 suppresses phosphorylation of STAT3 and Hp
expression……………………………………………………...22
5. Discussion…………………………………………………………24
6. Figure……………………………………………………………...29
7. Supplyment Data………………………………………………….. 47
8. Reference…………………………………………………………..55













List of Figures
Figure.1 The Haptoglobin (Hp) polymorphism in HepG2, FaDu and
SCC4 cell lines…………………………………………29
Figure.2. Effect of IL-6 induced on Hp production by HepG2, FaDu
and SCC4 cell lines…………………………………….30
Figure.3-1 The Hp regulated by IL-6- cytokines involving STAT3
signal stimulation in HepG2 cell lines…………………32
Figure.3-2 The Hp regulated by IL-6- cytokines involving STAT3
signal stimulation in FaDu and SCC4 cell lines…………33
Figure. 4-1-1 The pSTAT3 expression pretreatment with WP1066 in
HepG2 cell lines.………………………………………...35
Figure. 4-1-2 The Hpα2 and β expression pretreatment with WP1066 in
HepG2 cell lines.………………………………………..37
Figure. 4-2-1 The pSTAT3 expression pretreatment with WP1066 in
FaDu cell lines.………………………………………….39
Figure. 4-2-2 The Hpα2 and β expression pretreatment with WP1066 in
FaDu cell lines.………………………………………….41
Figure. 4-3-1 The pSTAT3 expression pretreatment with WP1066 in
SCC4 cell lines.…………………………………………43
Figure. 4-3-2 The Hpα2 and β expression pretreatment with WP1066 in
SCC3 cell lines.…………………………………………45
Figure S1. Domestic OCSS lines to survey Hp polymorphism……47
Figure S2. Determine the concentration of IL-6 in OCSS lines…...48
Figure S3. Subcellular fractionation to prove the pSTAT3 in the
Nucleus…………………………………………………49

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