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系統識別號 U0026-2608202016540900
論文名稱(中文) 探討登革病毒對甲狀腺癌細胞影響
論文名稱(英文) Dengue virus infection in thyroid cancer cells
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 108
學期 2
出版年 109
研究生(中文) 藍祥榮
研究生(英文) Hsiang-Rong Lan
學號 S46074158
學位類別 碩士
語文別 英文
論文頁數 53頁
口試委員 指導教授-彭貴春
口試委員-黃士銘
口試委員-簡玉雯
口試委員-羅玉枝
中文關鍵字 甲狀腺癌  癌幹細胞  登革病毒  登革病毒受體  膜蛋白CD44 
英文關鍵字 thyroid cancer  cancer stem cells  dengue virus  receptors for DENV  CD44 
學科別分類
中文摘要 甲狀腺癌是最常被診斷的內分泌癌,其所導致的死亡人數比其他所有內分泌癌的總和還多。大多數甲狀腺癌術後預後良好,但仍會有復發和轉移的情況。而癌幹細胞就是被認為是導致甲狀腺癌復發的主因,但至今尚未有人對於甲狀腺癌症中的癌幹細胞有深入的研究及討論。從我們先前的研究發現骨髓及肝癌中的癌幹細胞都很容易受登革病毒的感染。我們也將取得的甲狀腺癌檢體(包含腫瘤及非腫瘤組織)分別進行登革病毒感染,同時以流式細胞術去檢測其幹細胞族群比例。結果顯示,甲狀腺癌細胞也非常容易受登革病毒感染,且腫瘤組織受登革病毒感染後所產生的病毒效價也高於非腫瘤組織。有趣的是,我們發現腫瘤組織又可以分成易受登革病毒感染以及不易受登革病毒感染兩群。然而,進一步去檢測這兩群細胞中,癌幹細胞的族群表現,結果並無差異。從實驗室先前的研究顯示,膜蛋白CD44可能是登革病毒感染皮膚幹細胞的重要受體。所以我們接著也去檢測CD44在甲狀腺癌幹細胞中的表現,結果顯示有CD44表現的癌幹細胞與登革病毒感染所產生的病毒效價有顯著正相關。接著我們也利用玻尿酸,一種CD44的受體,去阻礙甲狀腺癌細胞中的CD44,然後再以登革病毒感染,並檢測病毒感染情況。結果顯示,利用玻尿酸阻礙CD44並不會影響登革病毒感染甲狀腺癌細胞。因此我們猜測,或許CD44與登革病毒之間的關聯只有在癌幹細胞上。由於甲狀腺檢體的細胞太稀少,所以我們選用高度表現CD44的甲狀腺癌細胞株MDA-T85細胞接續研究CD44與登革病毒在甲狀腺癌中的關聯。我們再次利用玻尿酸去阻礙MDA-T85細胞及其癌幹細胞中的CD44,再以登革病毒感染,檢測感染情況,結果顯示,以玻尿酸去阻礙癌幹細胞中的CD44對登革病毒感染有些微抑制的情況,但在整體的細胞中並無差異。為了更直接去研究CD44參與在登革病毒感染的角色,我們利用特製的小分子干擾核糖核酸去降低細胞中CD44的表現,接著以登革病毒去感染細胞。結果顯示,利用小分子干擾核糖核酸抑制MDA-T85細胞及其癌細胞中CD44的表現對於登革病毒感染都確實有抑制的效果。總結以上,甲狀腺癌細胞容易受登革病毒感染,且CD44可能是作為登革病毒進入甲狀腺癌細胞之受體。
英文摘要 Thyroid cancer is the most frequently diagnosed endocrine cancer and causes more deaths than all other endocrine cancers combined. Most thyroid cancers represent good prognosis after surgery, but sometimes the recurrence and metastasis are still occurring. The existence of cancer stem cells (CSCs) has been found to be correlated with the recurrence of thyroid cancers. The properties of CSCs in thyroid cancers have not been thoroughly discussed before. Our recent works on bone marrow and hepatocellular carcinoma (HCC) have shown that the CSCs population can be infected by dengue virus (DENV) efficiently. In the preliminary data, the tissues from tumorous or non-tumorous of thyroid cancer patients were utilized in the current research. Cells obtained in these tissues were subjected to DENV infection as well as FACS analysis was applied to evaluate the levels of cancer stem cell populations in these tissues. The result showed that the cells from thyroid tumors were highly permissive to DENV infection, and the viral titers were significantly higher than those from the non-tumor tissues. Interestingly, we found the cells from thyroid tumor tissues had two different infected performance, permissive and non-permissive. However, the expressions of CSC populations in DENV permissive and non-permissive group were no different. Therefore, we wanted to investigate the characteristics of CSCs in thyroid cancer during DENV infection. Previously, our lab found that CD44 played an important role as receptor for DENV infection in skin stem cells. We then evaluated the expression of CD44 in thyroid CSCs prior to DENV infection. The result suggested that the populations of stem cells in tumor tissues with CD44 were significantly positively correlated with DENV titers. Hence, we used hyaluronic acid (HA), a natural ligand for CD44, to do CD44 blocking assay in thyroid cancer cells. The results showed that blocking of CD44 would not affect the viral titers during DENV infection. Therefore, we speculated that the correlation may only specific in CSCs. However, the primary cells which isolated from thyroid cancer specimen were very rare. In order to testify the CD44 plays a role in thyroid cancer stem cells during DENV infection, we used MDA-T85 cells that is a thyroid cancer cell line for the purpose. MDA-T85 cells expressed high levels of CD44 by FACS analysis, followed by blocking assay with HA. The results suggested that blocking of CD44 in whole cells of MDA-T85 cells would not affect the virus titers during DENV infection, but an opposite outcome of CSCs of MDA-T85 cells was observed. To further confirm the role of CD44 involved in DENV infection, we used siRNA sequences to knockdown CD44 gene in MDA-T85 cells before DENV infection. The results displayed that silencing of CD44 by siRNA could suppress DENV infection in both whole cells and CSCs of MDA-T85 cells. In summary, the primary cells of thyroid cancer specimens are permissive to DENV and CD44 may be a potential receptor for DENV infection in thyroid cancer cell.
論文目次 中文摘要 I
Abstract III
Acknowledgement V
Table of contents VII
List of Figures X
Abbreviation Index XI
Introduction 1-7
1. Thyroid cancer 1
2. Treatment resistance and prognosis in thyroid cancer 1
3. Cancer stem cells (CSCs) effect Metastasis and prognosis 2
4. The origin of the CSCs 3
5. The common markers of CSCs 3
6. Dengue virus (DENV) Epidemiology and pathophysiology 4
7. Receptors for DENV 5
8. The structure and functions of CD44 5
9. The role of CD44 in cancer cells 6
10. Hypothesis 7
Materials and Methods 8-25
A. Materials 8
1. Cell lines and virus 8
2. Antibody (thyroid cancer panel) 8
3. Antibody (isotype) 8
4. Magnetic beads 9
5. Medium and Reagent 9
6. Plastic and glass equipments 11
7. Instruments and machines 13
8. Primer 14
9. siRNA 14
10. Ingredients in buffer and medium 15
B. Methods 17
1. Cell line culture (BHK-21, Vero and MDA-T85) 17
2. Thyroid cancer specimens 17
3. Multicolor FACS analysis 18
4. Dengue virus expansion 18
5. Ex vivo and in vitro infection of DENV 19
6. Magnetic Bead isolation 20
7. Ex vivo and in vitro blocking assay of Hyaluronic acid (HA) 20
8. RNA interference assay with siRNA 21
9. qPCR 22
10. Western blot 23
11. Plaque assay 24
12. Statistical analysis 25
Results 26-30
1. Tumor cells of thyroid cancer patients were highly permissive to DENV infection. 26
2. Cell populations of cancer stem cells were no difference between DENV permissive and non-permissive groups. 27
3. The percentage of CD44 in stem cells of tumor tissues was significantly positively correlated with DENV titers 27
4. Blocking of CD44 with HA did not inhibit the infection of DENV in thyroid cancer cells. 28
5. MDA-T85, a thyroid cancer cell line containing high percentage of CD44+ cell population, were permissive to DENV infection. 29
6. Blocking CD44 with HA could inhibit the infection of DENV in cancer stem cell of MDA-T85 cells. 29
7. Inhibition of CD44 by siRNA could suppress DENV infection in both whole cells and cancer stem cells of MDA-T85 cells. 30
Discussion 31-35
References 36-40
Figures 41-53
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