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研究生:呂萱萱
研究生(外文):Hsuan-Hsuan Lu
論文名稱:經檳榔處理之口腔纖維母細胞可促進口腔上皮細胞之癌化
論文名稱(外文):Areca-treated fibroblasts enhance tumorigenesis of oral epithelial cells
指導教授:張國威
指導教授(外文):Kuo-Wei Chang
學位類別:博士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2008
畢業學年度:97
語文別:英文
論文頁數:88
中文關鍵詞:檳榔口腔纖維母細胞老化癌化口腔癌
外文關鍵詞:areca (betel)oral fibroblastssenescencetumorigenesisoral cancer
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在亞洲地區約有數億人口有嚼食檳榔的習慣,因此在這些地區嚼食檳榔與口腔癌的形成有高度相關性,但檳榔子水萃取物 (areca nut extract) 對口腔細胞之致病分子機轉仍未釐清。嚼食檳榔除了與口腔相關疾病及癌化相關外,亦會增加口腔相關之炎性疾病之病變。細胞外基質金屬蛋白酶 (matrix metalloprotease;MMP) 表現之變異亦與口腔疾病之病理機轉相關。本研究探討檳榔子水萃取物
對口腔纖維母細胞之可能影響,進而對口腔病變的後續影響。
本研究之假設為經檳榔暴露之間質細胞可能影響口腔癌化之進行。於本研究中發現長期且低劑量之檳榔子水萃取物處理 (小於10 μg/ml) 會誘發口腔纖維母細胞之提早老化。在這些老化的口腔纖維母細胞中除了表現出生長停滯的現象外,也伴隨老化相關半乳糖水解脢 (senescence-associated β-galactosidase;SA-β-Gal) 的表現與其他老化相關表型之顯現。在檳榔子水萃取物誘發老化之口腔纖維母細胞 (ANE-induced senescent oral fibroblasts;ASOF) 中 MMP-2 表現量及活性有顯著上升的現象。將口腔癌細胞株處以 ASOF 的培養液 (supernatants of ASOF) 後發現除了會活化 AKT 訊息傳遞路境外,也觀察到細胞增生、爬行與非貼附性增生之轉型表型均有顯著上升。ASOF 培養液的促進效果會被 MMP-2 或是 AKT 的抑制劑所阻礙。而合併 ASOF 之細胞植入更會促進口腔癌細胞株在裸鼠體內形成腫瘤之能力。此外、ASOF 培養液亦會促進噬中性白血球在體外的侵襲能力。本研究的結果可結論檳榔子水萃取物對口腔纖維母細胞之影響,以及其後透過分泌性分子改變微環境以影響口腔上皮癌化的過程。如此可強化檳榔子水萃取物的致癌證據並且有助於進一步阻斷口腔癌之進行。
Several hundred million Asians chew areca nut, which is highly associated with the oral carcinogenesis in people of this region. The impacts of areca nut extract (ANE) on oral target cells are largely unclear. Areca nut chewing is associated with an increase in the incidence of oral neoplastic or inflammatory diseases. Aberrations in matrix metalloprotease (MMP) expression are associated with the pathogenesis of oral diseases.
This study investigated the potential effects of ANE on human oral fibroblasts and the consequential impacts on pathogenesis. This study hypothesized an inductive role of areca nut-exposed stromal cells in the progression of oral carcinomas. Chronic subtoxic (less than 10 μg/ml) ANE
treatment resulted in premature growth arrest, the appearance of senescence-associated β-galactosidase activity and various other senescence-associated phenotypes in oral fibroblasts. Oral fibroblasts established from
older individuals had a higher propensity to become ANE-induced senescent oral fibroblasts (ASOF). An activation of MMP-2 was identified in ASOF. The supernatants of ASOF activated AKT signaling pathway in oral carcinoma cells. The
enhancement of proliferation, migration and anchorage-independent growth of oral carcinoma cells elicited by such supernatants can be abolished by blockers against MMP-2 or AKT. Subcutaneous co-injection of ASOF into nude mice significantly enhanced the tumorigenecity of xenographic oral carcinoma cells. The supernatants also enhanced the invasion of neutrophil in an in vitro model. This study concludes that ANE may impair oral fibroblasts and then modulate the microenvironment that dives the progression of oral epithelial oncogenesis via their secreted molecules, such as MMP-2.
LIST OF ABBREVIATIONS.....................................11
CHAPTER I. INTRODUCTION...................................12
Section A. Human oral cancer and areca (betel) chewing....13
A.1. Oral cancer..........................................13
A.2. Areca nut............................................14
Section B. Cellular senescence and cell cycle.............15
B.1. Cellular senescence..................................15
B.2. Senescent phenotypes.................................16
(a) Stress granules.......................................17
(b) p53 tumor suppressor..................................17
(c) p21WAF1/CIP1/SDI1.....................................18
(d) p16INK4a/Rb...........................................18
Section C. Extracellular matrix (ECM) and MMP-2...........19
C.1. Extracellular matrix.................................19
C.2. Matrix Metalloproteinases (MMP) and MMP-2............20
Section D. Hypothesis.....................................21
CHAPTER II. MATERIALS AND METHODS.........................23
A. Materials..............................................24
B. Anchorage-independent growth...........................24
C. Cell culture, growth curve.............................25
D. ELISA assay............................................26
E. Flow cytometry.........................................26
F. Gelatin zymography.....................................26
G. Invasion assay.........................................27
H. Migration assay........................................28
I. RT-PCR.................................................28
J. Senescence-associated ß-galactosidase assay............29
K. Tumorigenesis..........................................29
L. Western blot analysis..................................30
M. Statistics.............................................30
CHAPTER III. RESULTS......................................31
Section A. ANE-induced premature senescence in oral fibroblasts and regulated MMP-2, TIMP-1 and u-PA mRNA expression and up-regulated MMP-2 activity................32
A.1. Chronic subtoxic ANE treatment resulted in the premature growth arrest of oral fibroblasts...............32
A.2. p16, p21 and Rb expression in oral fibroblasts following continuous treatment with ANE...................34
A.3. Chronic subtoxic ANE treatment induced MMP-2 expression of oral fibroblasts............................35
Section B. ANE-treated oral fibroblasts enhance the proliferation and migration via the production of MMP-2, and induce tumorigenesisin oral cells.....................37
B.1. Supernatants of ASOF enhanced the proliferation and migration of oral carcinoma cells through MMP-2 activity..37
B.2. Supernatants of ASOF activated AKT signals in oral carcinoma cells...........................................38
B.3. Supernatants of ASOF enhanced anchorage-independent growth of oral carcinoma cells............................38
B.4. Supernatants of ASOF enhanced anchorage-independent growth of OECM-1 cells....................................39
B.5. ASOF enhanced the tumorigenicity of oral carcinoma cells.....................................................40
B.6. Histopathology of xenographic tumors and the characteristics of cultivated cells from xenographic tumors ...................................................41
Section C. Areca nut extract-treated oral fibroblasts modulate the invasiveness of polymorphonuclear leukocytes via the production of MMP-2...............................42
CHAPTER IV. DISCUSSION....................................44
CHAPTER V. FIGURES........................................53
Figure 1 Occurrence of growth arrest in fibroblasts following ANE treatment...................................54
Figure 2 Appearance of Senescent-associated β-alactosidase activity in oral fibroblasts following ANE treatment......55
Figure 3 The responses of oral fibroblasts to ANE as related to the age of individuals providing the tissue....56
Figure 4 Senescence phenotypes in representative ASOF.....57
Figure 5 p16, Rb and p21 expression in oral fibroblasts with the treatment of ANE.................................58
Figure 6 ANE up-regulated the expression level of ECM-associated genes in oral fibroblasts......................59
Figure 7 MMP-2 protein expression and activity in oral fibroblasts following ANE treatment.......................60
Figure 8 Supernatants of ASOF increased the proliferation of oral carcinoma cells...................................61
Figure 9 Supernatants of ASOF increased the migration of oral carcinoma cells......................................62
Figure 10 MMP-2 activity in the supernatants from ASOF....63
Figure 11 Supernatants of ASOF enhanced migration of oral squamous carcinoma cells through MMP-2 activity...........64
Figure 12 Supernatants of ASOF activated AKT in oral carcinoma cells...........................................65
Figure 13 Supernatants of ASOF enhanced the anchorage-independent growth of SAS cells...........................66
Figure 14 Supernatants of ASOF enhanced the anchorage-independent growth of OECM-1..............................68
Figure 15 The supernatants of ASOF influence on the
anchorage-independent growth of OC3 cells.................70
Figure 16 Co-injection of ASOF enhanced the tumorigenesis of SAS cells..............................................71
Figure 17 Co-injection of ASOF enhanced the transient tumorigenesis of OECM-1 cells.............................72
Figure 18 Histopathology of xenographic tumors............73
Figure 19 The supernatants of ASOF affects the invasiveness of PMN leukocytes.........................................74
CHAPTER VI. TABLES........................................75
Table 1. Primers used in the present study................76
Table 2. Antibodies used in the present study.............77
Table 3. Schematic diagram of the transwell apparatus and the invasiveness of PMN leukocytes........................78
CHAPTERVII. APPENDIX......................................79
Appendix 1 MMP-2 (gelatinase A) and MMP-9 (gelatinase B) activity in areca-treated oral fibroblasts................80
Appendix 2 MMP-2 expression and activity in oral carcinoma cells.....................................................81
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