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系統識別號 U0026-2008201511214400
論文名稱(中文) 登革熱病毒感染人類肝腫瘤CD133+CD61+前驅細胞
論文名稱(英文) Cells with CD133+CD61+ phenotypes are highly permissive to dengue virus infection
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 103
學期 2
出版年 104
研究生(中文) 蔡明哲
研究生(英文) Ming-Che Tsai
學號 S46024072
學位類別 碩士
語文別 英文
論文頁數 54頁
口試委員 指導教授-彭貴春
口試委員-張志鵬
口試委員-謝清河
中文關鍵字 登革熱病毒  肝癌  癌幹細胞  幹細胞  前驅細胞 
英文關鍵字 dengue virus  hepatoma  cancer stem cells  stem cells  progenitor cells 
學科別分類
中文摘要 人類肝惡性腫瘤(Hepatocellular carcinoma, HCC)是世界上常見的疾病之一,並且每年在全世界皆有死亡案例發生。近期,癌幹細胞的理論已經被指出並且在腫瘤組織中被揭露,這些癌幹細胞具有正常幹細胞的生物特性,如自我增生再造,高度分化能力等,並且與一些幹細胞與前驅細胞相近。然而,癌幹細胞的表現形態非常多,定義癌幹細胞亦不容易。本篇研究首先利用登革熱病毒會感染人類骨髓造血幹細胞或前驅細胞的特性,藉此假設:若癌幹細胞具有與骨髓幹細胞有相同特性的話,則登革熱病毒可以藉由高度病毒專一性標的感染。本研究使用人類肝癌組織,經由獲准的IRB程序進行實驗,每項檢體由同一個病人經由醫師臨床診斷,分辨出正常肝組織與惡性腫瘤組織進行後續實驗,並使用研磨或酵素方式取得細胞懸浮液,細胞經由計算過後以1 MOI感染,並且以病毒斑測試觀察動態病毒複製的變化,以及正常與惡性腫瘤組做比較;生物特性藉由西方墨漬法觀察。在流式細胞儀分析中初步揭露在肝癌組織中的巨核前驅細胞為主要被登革熱病毒感染的標的,並且後續使用磁珠分選方式可以證實此CD133+或CD61+細胞群能支持病毒感染複製;不含有此細胞群的肝癌細胞則相對感染能力低。此外,由肝癌組織細胞感染產生之登革熱病毒也不表現病毒外殼結構,顯示此病毒感染不同於細胞株之特性。我們也許可以藉由此種方式製造出抗癌疫苗;或者新穎標靶藥物去消去惡性腫瘤細胞,達到癌症治療效果。
英文摘要 Human hepatocellular carcinoma (HCC) is a common disease worldwide, causing many deaths annually. Recently, the theory of cancer stem cells (CSCs) has been proposed and investigated for the presence of the CSCs in tumor/cancer tissues. The characteristics of these CSCs, such as the ability of self-renewing, capability of differentiation, and sustaining the propagation specifics similar to the conventional stem cells, entitle them to be closely related to stem/progenitor cells. However, the phenotypes of the CSCs remain at large. In this study, we took the advantages of the infectability of dengue virus (DENV) to human bone marrow (HBM) hematopoietic stem/progenitor cells. We therefore hypothesized that DENV can infect the CSCs if these cells share the phenotypes of HBM hematopoietic stem/progenitor cells. Specimens were obtained from hepatoma patients with approved IRB protocol. Each tissue was divided into tumor and normal parts based upon physiological diagnosis. Single cell suspension was prepared from tissues that were enzymatic digested of connective materials in tissues. Cell density at 1×106 cells per tube per time point was utilized for the kinetic studies, either infected with DENV at 1 MOI (multiplicity of infection) or uninfected as the control. Viral titers in the supernatant were evaluated by the plaque assay, while biological elements of cells were performed by western blot analysis. The results demonstrated that high DENV viral titers were dominantly from the cells in the tumor part, compared to that of the normal part from the same donor. FACS analysis revealed that megakaryocytic lineage cells in hepatoma tissue were the dominant cell populations targeted by DENV. Furthermore, isolation of cells with surface markers of CD133+ and/or CD61+ in hepatoma proved to be highly permissive to DENV infection, in contrast to the cells with surface markers of CD61- and CD133- which appeared not to be infectable. Biological properties of the virus produced from the supernatants of the DENV infected hepatoma cells revealed that DENV produced from infected primary hepatoma cells had no capsid protein as compared with virus derived from the DENV infected Vero cells. The cumulative results suggested that the permissive cells solely presented in tumor tissue could be the CSCs. We will expand our views to other types of cancer tissue to address if they have the same phenomena as HCC. This could pave a new avenue for anti-cancer vaccine and drug development or targeting strategies.
論文目次 Chinese Abstract I
Abstract II
Acknowledgement IV
Table of Contents V
Table List VIII
Figure List IX
Introduction 1
Materials and Methods 6
IRB Approval 6
Extraction of Primary Human Hepatocellular Carcinoma Cells (HCCs) 6
Cell Viability Test of Cultured-HCCs 7
Cell Culture 7
HCCs Infected with DENV 8
DENV Infectivity Test of Suspension and Adhesion HCCs 8
Dengue Virus Plaque Assay 8
Immunohistochemistry Stain (IHC) 9
Multi-color FACS analysis 10
Magnetic Beads Sorting 10
Amplification of Dengue Virus 11
Purification of Dengue Virus 11
Western Blotting 12
Electron Microscopy 13
Statistical Analysis 13
Results 14
1. Bone marrow cells were permissive to dengue virus infection 14
2. Human hepatocellular cells were permissive to dengue virus infection 14
3. IHC stain was showed that only tumor part can be infected with dengue virus 15
4. Undifferentiated hepatoma tumor cells can be infected with dengue virus 16
5. Multi-color flow cytometry analysis showed that dengue virus dominantly targeted to CD133+CD61+CD41+ megakaryocytic lineage cells 16
6. Magnetic beads sorted out CD133+CD61+ megakaryocytic lineage cells for dengue virus infection 17
7. Unconventional dengue virus morphology was observed by western blot and electron microscopy 17
Discussion 19
References 24
Tables 29
Table 1. Gating strategy of multi-color FACS analysis. 29
Table 2. Eight-color FACS stem cell-associated markers analysis results. 31
Table 3. Clinical data of hepatoma patients 43
Table 4. The relationship between viral titer and patient properties 45
Figures 46
Figure 1. Human bone marrow cells were highly permissive to DENV replication. 47
Figure 2. Hepatoma normal and tumor part cells culture viability test. 48
Figure 3. Primary hepatoma tumor cells were permissive to DENV2 replication. 49
Figure 4. IHC staining of primary hepatoma cells in tumor part. 50
Figure 5. Only suspension hepatoma cells were infectable by DENV 51
Figure 6. Multi-color FACS analysis of DENV targeting cells 52
Figure 7. Direct isolation of potential permissive cells showed that the main DENV replicating cells were CD61+CD133+. 53
Figure 8. Unconventional dengue viral particles produced from hepatoma cells. 54
Figure 9. Special phenotype particle of DENV produced by hepatoma and demonstrated by electron microscopy. 55
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