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研究生:陳昌佑
研究生(外文):Chang-Yu Chen
論文名稱:Annexin A2蛋白在鼻咽癌中調控多種惡化表型和治療上的耐受性
論文名稱(外文):Annexin A2 regulates multiple malignant phenotypes and therapeutic tolerance in nasopharyngeal carcinoma
指導教授:陳建和陳建和引用關係
指導教授(外文):Chien-Ho Chen
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗暨生物技術學系所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:64
中文關鍵詞:鼻咽癌Annexin A2EMT化療放射性治療
外文關鍵詞:nasoparyngeal carcinoma (NPC)Annexin A2chemotherapyradiotherapyepithelial-mesenchymal transition (EMT)
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鼻咽癌細胞為一種位於鼻咽的惡性上皮細胞癌,其盛行於台灣及東南亞地區。不同於其他頭頸癌,鼻咽癌早期就具有高轉移能力而且晚期之病患,常因其高度侵犯轉移之特性,造成治療之失敗。因此更進一步了解相關分子,將有助於整體治療之成效。先前透過免疫組織化學染色法(IHC),發現annexin A2蛋白在鼻咽癌病人的腫瘤組織中有較高的表現量,近幾年不同腫瘤研究中也指出annexin A2蛋白質表現與腫瘤惡化相關性,因此進而探討annexin A2在鼻咽癌所扮演的角色。透過基因靜默(gene silence)抑制annexin A2表現,發現抑制annexin A2會降低癌細胞的生長能力,增加細胞對化療藥物 (Cisplatin、5-FU、Vincristine、 Docetaxel)與放射線治療之敏感性。抑制Annexin A2表現會降低細胞移行、黏附和侵犯能力。動物實驗也證實降低annexin A2表現會抑制腫瘤細胞的生長。進一步探討相關調控分子,發現annexin A2會調控上皮-間質細胞轉化(EMT)之相關分子之表現、AKT分子表現與磷酸化現象。在臨床層面分析,發現annexin A2高度表現於晚期鼻咽癌病患且與表現量與N期有相關性。藉由這些研究結果,我們更加了解annexin A2在鼻咽癌細胞的功能,未來annexin A2也許可以當成臨床上治療的一個標的與預後指標。

Nasopharyngeal carcinoma (NPC), originated from the epithelium of the nasopharynx, is a common malignant tumor. NPC mainly occurs in the Southeast Asia including Taiwan. Characteristically, NPC differs from other head and neck carcinomas, especially for its high metastasis character and poor efficiency of clinical treatment. Recently, many reports have indicated that annexin A2 might regulate the metastasis on different kinds of cancer. However, the tumorigenic function of annexin A2 in NPC is not yet understood. Annexin A2 shRNAs were used to evaluate the effects of annexin A2 suppression on NPCs. Annexin A2 silencing reduces the cell proliferation ability and increase chemotherapeutic drugs (Cisplatin, 5-FU, Vincristine and Docetaxel) and irradiation sensitivity. Moreover, annexin A2 not only up-regulates cell adhesion, migration, and invasion abilities on NPCs, but also involves in epithelial-mesenchymal transition (EMT). These cellular results were confirmed using tumor xenografts in mice, as annexin A2 silencing led to suppress tumor growth. Clinicl study reveals that the high level of annexin A2 on late-staged NPC patient tissues and associated with N stage. In summary, annexin A2 not only regulates many malignant phenotypes such as migration, invasion, adhesion and EMT, but also controls therapeutic tolerance in NPCs. Annexin A2 may serve as
prognostic markers and targets in NPC.

中文摘要 v
Abstract vi
Abbreviation vii
Acknowledgement…………………………………………...viii

1. Introduction 1
1.1 Nasopharyneal carcinoma (NPC)……………...…………………1
1.2 Chemo- and radiotherapy for nasopharyngeal carcinoma……..…3
1.3 Tumor-induced immune suppressive response…………………..3
1.3.1 Dendritic cell (DC) and DC-SIGN………………………4
1.4 Identify the DC-SIGN interacted protein: annexin A2…………..5
1.41 Annexin A2………………..………………………………6
1.42 The effect of annexin A2 in nasopharyngeal carcinoma…8
1.5 Epithelial to mesenchymal transition (EMT)……………………8
1.5.1 TGF-β induced EMT……………………………………10
1.5.2 TNF-α induced EMT……………………………………11
1.6 Akt regulating pathways related to cell proliferation, exocytosis
and irradiation resistance………………………………………..12

2. Aim of study 14

3. Materials and Methods
3.1 Cell and Culture………………………………………………..15
3.2 Patient’s tissue sample…………………………………………..15
3.3 Establishment of annexin A2-silenced NPC……………………16
3.4 Western blotting………………………………………….……16
3.5 RT-PCR…………………………………………………………17
3.6 Real-time PCR…………..………………………………………18
3.7 Cell proliferation and cytotoxicity assay………………………18
3.8 Clongenic assays for irradiation …………...............………...…19
3.9 Migration…………..……………………………………………19
3.10 Invasion………………………………………………………..19
3.11 Adhesion………………………………………………………20
3.12 Tube formation assay…………………………………………20
3.13 Immunofluorescence stain……………………………………20
3.14 Xenograft tumor mice model (SCID)…………………………21
3.15 H&E stain……………...………………………………………21
3.16 Immunohistochemistry……...…………………………………22
3.17 Statistic……………...…………………………………………22

4. Results
4.1 Knockdown of annexin A2 inhibits cell proliferation in NPC
cell lines…………..……………………………………………23
4.2 Annexin A2 involves in the resistance of therapeutic reagents
(cisplatin, 5-FU, vincristine and docetaxel)……………….24
4.3 Annexin A2 involves in the resistance of irradiation……...……24
4.4 Annexin A2 promotes cell migration, invasion, vascular
formation, and adhesion of nasopharyngeal carcinoma……...…25
4.5 Expression of annexin A2 induces an epithelial-mesenchymal
transition by activation of TGF-β pathway……………………..26
4.6 Annexin A2 promotes nasopharyngeal carcinoma growth
in vivo………………………………………………………….28
4.7 Expression of annexin A2 in human nasopharyngeal carcinoma
tissues…………………………………………………………..28

4.8 Annexin A2 regulates Akt expression in NPC………………….29

5. Conclusion 30

6. Discussion 31

Appendix
A.1 Certificate of TMU-JIRB Approval 35

List of References 36
Figures 41


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