系統識別號 U0026-2308201318103400
論文名稱(中文) 探討Calprotectin在口腔癌及基質細胞所扮演的角色
論文名稱(英文) Studying the Role of Calprotectin in Oral Cancer and Stromal Cells
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 101
學期 2
出版年 102
研究生(中文) 陳怡雯
研究生(英文) Yi-Wen Chen
學號 T16004043
學位類別 碩士
語文別 英文
論文頁數 50頁
口試委員 指導教授-吳梨華
中文關鍵字 S100A8  S100A9  口腔癌  血管新生 
英文關鍵字 S100A8  S100A9  oral cancer  angiogenesis 
中文摘要 在台灣口腔癌是常見的男性癌症之一。Calprotectin是由S100A8及S100A9這兩個蛋白質所形成的異型二聚體。這兩個蛋白質是屬於可與鈣離子結合的S100蛋白質家族成員,主要是表現在一些骨隨細胞。Calprotectin 除了表現在與發炎相關的細胞外,在許多不同類型的癌症中也發現calprotectin的存在。最近也發現,在急性及慢性發炎中,calprotectin可作為發炎前期的指標。因此,calprotectin在與發炎相關的癌症中扮演一個相當重要的角色,其中也包含了口腔癌。雖然目前關於calprotectin的功能已有一些研究,但其在口腔癌中所扮演的角色目前還並不清楚。根據我們實驗室之前針對口腔癌病人檢體做的免疫組織化學染色切片顯示:在腫瘤周邊的基質細胞內,其S100A9的表現量上升,與病人有較差的預後呈正相關。本篇研究主要的目的是要探討calprotectin在口腔癌細胞、內皮細胞及單核球中所扮演的角色。其中,內皮細胞及單核球富含於腫瘤基質中。利用免疫螢光染色,我們發現calprotectin不只表現在腫瘤細胞中,也表現在腫瘤周邊的基質細胞中,其中包含了表現CD15的嗜中性白血球,以及表現CD11b和CD68的單核球或巨噬細胞。這個結果表示calprotectin對於口腔癌可能同時具有自體分泌及旁分泌的作用。雖然改變calprotecctin在細胞內的表現量對於細胞生長及入侵的能力在不同的細胞株而有不同的結果,但對於細胞的爬行能力扮演著促進的角色。除此之外,由口腔癌細胞分泌的calprotectin能抑制抗發炎因子IL-10的產生,並促進血管新生。與in vitro實驗結果相符,S100A9具有促進腫瘤在老鼠體內生長的能力。最後總結,calprotectin,特別是S100A9,在口腔癌形成過程中扮演促癌的角色。了解calprotectin在口腔癌細胞及基質細胞中所扮演的角色對於未來研發新的口腔癌的治療方法會有所幫助。
英文摘要 Oral cancer is one prevalent male cancer type in Taiwan. Calprotectin is a heterodimer consisting of S100A8 and S100A9. Both proteins belong to a large calcium-binding S100 family and are predominantly expressed by myeloid cells. In addition to their expression in inflammatory cells, calprotectin has been detected in many cancer types and has recently emerged as a pro-inflammatory marker for both acute and chronic inflammatory diseases. Taken together, calprotectin may play a prominent role in inflammation-associated cancer including oral cancer. Although a number of putative functions have been proposed for calprotectin, its biological role particularly in oral cancer remains poorly defined. Using immunohistochemistry staining, we were able to find that the increase of S100A9, a subunit of calprotectin, in the stromal tissue adjacent the tumor tissues was significantly associated with poor clinical outcome. The purpose of this study is to investigate the role of calprotectin in oral cancer cells, endothelial cells and monocytes. The latter two types are rich in tumor stroma. Immunofluorescence microscopy showed that calprotectin was not only detected in oral cancer cells but also in their surrounding stromal cells including monocytes or macrophages expressing CD68+ or CD11b+, and neutrophils expressing CD15+ cells, suggesting the possibility of autocrine and paracrine effect of calprotectin on oral tissues. Although altering the expression of calprotectin affected cell proliferation and invasion in cell type-dependent manner, calprotecin functioned as a promoting molecule for cell migration. Moreover, calprotectin secreted from oral cancer cells inhibited anti-inflammatory IL-10 production and acted as positive regulator in angiogenesis. S100A9 manifested a more potent tumor promoting effect in vitro. Consistent with the notion, S100A9 promoted tumorigenesis in vivo. Together, stromal calprotectin especially S100A9 may play an oncogenic role in oral carcinogenesis. Understanding the potential role of calprotectin in oral cancer and stromal cells will facilitate the development of new therapeutic approaches for oral cancer treatment.
論文目次 中文摘要 I
Abstract II
致謝 IV
Content VI
Table, figure and appendix contents IX
Abbreviations XI
I. Introduction 1
1-1 Oral cancer 1
1-2 Cancer cells and their microenvironment 1
1-3 Calprotectin 2
1-4 Calprotectin in stroma and cancer 2
II. Specific aims 5
III. Materials and methods 6
3-1 Materials 6
3-2 Cell culture 7
3-3 Construct pcDNA3.1A-S100A8 or S100A9 plasmids 8
3-4 Establish S100A8 or S100A9 overexpressing stable clones 8
3-5 Establish S100A8 or S100A9 knockdown cells 8
3-6 Doubling time 9
3-7 Wound healing 9
3-8 Cell invasion assay 9
3-9 Prepare conditioned medium (CM) 10
3-10 Semi-quantitative reverse transcription-PCR (RT-PCR) 10
3-11 MTT assay 10
3-12 Migration assay 11
3-13 Tube formation assay 11
3-14 Western blot analysis 11
3-15 Immunofluorescence staining 12
3-16 Xenograft transplantation 12
IV. Results 13
4-1 Calprotectin was expressed in oral cancer cells and certain stromal cell types including monocytes/macrophages and neutrophils 13
4-2 Altered S100A8 or S100A9 expression affected oral cancer cell proliferation 13
4-3 S100A8 or S100A9 induced migration but inhibited invasion in oral cancer cells 14
4-4 S100A8 or S100A9 secreted from cancer cells inhibited the production of anti-inflammatory cytokine IL-10 15
4-5 S100A8 or S100A9 acted as positive regulator in angiogenesis 16
4-6 S100A9 promoted turmorigenesis in vivo 16
V. Discussion 18
References 21
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