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系統識別號 U0026-2608201910462400
論文名稱(中文) 不同活性之STIM1分子調控骨肉瘤細胞的爬行能力與黏著斑的變化
論文名稱(英文) Cell migration and focal adhesion in osteosarcoma are regulated by different STIM1 activities
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 107
學期 2
出版年 108
研究生(中文) 林懿莘
研究生(英文) Yi-Hsin Lin
電子信箱 wsp90020@gmail.com
學號 P86064182
學位類別 碩士
語文別 英文
論文頁數 69頁
口試委員 指導教授-邱文泰
口試委員-湯銘哲
口試委員-王仰高
口試委員-郭津岑
中文關鍵字 鈣離子  鈣池調控之鈣離子流  細胞爬行  黏著斑  STIM1分子 
英文關鍵字 Ca2+  SOCE  cell migration  focal adhesion turnover  FA  STIM1 
學科別分類
中文摘要 骨肉瘤普遍好發在青少年族群中,名列前幾名惡性的癌症之一,其中80-90 %的病人在使用化學治療之前會出現轉移的情形,因此,骨肉瘤細胞的轉移機制為癌症治療研究的重要議題。細胞爬行是癌症轉移不可或缺的機制,黏著斑的動態變化(組成和瓦解)為其重要的步驟,而由鈣池調控的鈣離子流可藉由提升細胞內鈣離子濃度並多種途徑調控黏著斑的變化。鈣池調控之鈣離子流為非興奮性細胞中最主要的細胞內鈣離子補充來源,當內質網內的鈣離子濃度降低,STIM1分子將會被活化,啟動鈣池調控之鈣離子流。先前研究發現癌細胞過度表達STIM1分子會促進轉移,然而其中的分子機制仍未被充分了解,而且此現象並沒有在骨肉瘤細胞中證實。在此論文研究中,我們利用表達不同活性之STIM1分子的骨肉瘤細胞,探討STIM1分子與細胞爬行、黏著斑變化之間的關係,並藉由全內反射螢光顯微技術,清楚觀察活化之STIM1分子在近細胞膜處形成的點狀結構以及細胞的黏著斑。我們在實驗中發現,表達持續活化STIM1分子的骨肉瘤細胞降低了爬行能力以及黏著斑的大小,但也同時增加了黏著斑的數量;在另一方面,表達不活化STIM1分子的骨肉瘤細胞則增加了爬行能力以及黏著斑的大小,但減少了黏著斑的數量。實驗結果顯示,表達持續活化STIM1分子的骨肉瘤細胞因增加鈣池調控之鈣離子流而增加m-calpain的活性,進而減少黏著斑的大小以及細胞爬行的能力。透過此研究,我們可以更了解調控癌細胞爬行的機制,為骨肉瘤轉移的治療提供貢獻。
英文摘要 Osteosarcoma is the most common primary malignant bone tumor in children and young adults, and most patients developed metastasis before they are diagnosed. This indicates that the prevention of cancer cell metastasis is a big issue in cancer therapy. Cell migration is essential for cancer metastasis, whereas focal adhesion (FA) turnover (assembly and disassembly) is the critical step in regulating the migration process. Calcium (Ca2+) is involved in the regulation of FA dynamics. Previous studies have shown that store-operated Ca2+ entry (SOCE) can promote FA turnover through several ways by elevating the cytosolic Ca2+. The most well-known and the major Ca2+ entry in non-excitable cells is SOCE, and STIM1 is the main protein that stimulates the SOCE in response to ER Ca2+ depletion in vertebrates. Overexpression of STIM1 proteins has been reported to be correlated with metastatic progression in various cancers. However, the molecular mechanisms underlying SOCE-mediated metastasis are still poorly understood and the phenomenon has not been investigated in the osteosarcoma cancer cell. In this study, we used various STIM1 variants (wild-type, constitutively activated or dominant-negative STIM1) overexpressing in human bone osteosarcoma U2OS cells to investigate the relationship between different STIM1 activities and cell migration. The juxta membrane STIM1 puncta and FAs could be clearly seen using TIRF microscopy. We found that U2OS cells overexpressed with constitutively active STIM1 decreased the migration ability of FAs, while increasing their number but decreasing their size. On the other hand, the dominant-negative STIM1 increased the migration ability of FAs while decreasing their number but increasing their size. Our results showed that the constitutively active STIM1 enhanced SOCE, which may increase the m-calpain activity, resulting in the small FAs and thus decreasing the cell migration. Through this study, the biological mechanism of cancer cell migration may be made clearer, which may contribute to investments in potential targets for osteosarcoma metastasis.
論文目次 Abstract III
中文摘要 V
Acknowledgement VI
Contents VII
Table contents IX
Figure contents X
Chapter 1 Introduction 1
1.1 Cell migration 1
1.1.1 Cell migration in cancer cell 1
1.1.2 Steps and components of cellular migration 1
1.2 Focal adhesion (FA) 3
1.2.1 The structure and components of FA 3
1.2.2 FA turnover and cell migration 4
1.3 The importance of Ca2+ 4
1.3.1 Ca2+ homeostasis 4
1.3.2 Stored-operated calcium entry (SOCE) 6
1.3.3 Ca2+ in FA turnover and cell migration 7
1.4 Motivation and specific aims 7
Chapter 2 Materials and Methods 9
2.1 Database research 9
2.2 Cell line and cell culture 9
2.3 Total internal reflection fluorescence microscopy and image analysis 11
2.4 Confocal microscopy 11
2.5 Single-cell Ca2+ measurement 12
2.6 Focal adhesion profiles under TIRF microscopy 12
2.7 Time-lapse recording of focal adhesion dynamic 13
2.8 Polarity assay 13
2.9 Wound healing assay 14
2.10 2D single-cell migration assay 14
2.11 Transwell assay 15
2.12 Calpain activity assay 15
2.13 Western blotting 16
2.14 Statistical analysis 17
Chapter 3 Results 18
3.1 Constitutively active STIM1 increases SOCE level 18
3.2 Constitutively active STIM1 decreases the migration ability 20
3.3 STIM1 variants regulated the pattern of FAs 21
Chapter 4 Discussion 25
References 29
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