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系統識別號 U0026-2708202010354500
論文名稱(中文) 登革病毒與茲卡病毒對神經幹細胞之影響
論文名稱(英文) Neural stem cells in dengue virus and zika virus infections
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) 郭俞菱
研究生(英文) Yu-Ling Kuo
學號 S46071053
學位類別 碩士
語文別 英文
論文頁數 46頁
口試委員 指導教授-彭貴春
口試委員-羅玉枝
口試委員-張科
口試委員-簡玉雯
中文關鍵字 登革病毒  茲卡病毒  癌幹細胞 
英文關鍵字 dengue virus  zika virus  cancer stem cells  CD133  CD44 
學科別分類
中文摘要 茲卡病毒屬於一種黃熱病毒,已知其能夠感染神經幹細胞及發育中的中樞神經前驅細胞。研究指出,與茲卡病毒同屬於黃熱病毒之登革病毒也能夠感染神經細胞,在某些案例中甚至發現可能造成腦炎。由於登革病毒與茲卡病毒在基因組成上十分相似,並且感染此兩種病毒的患者擁有共通的臨床症狀,例如:發燒、起紅疹、肌肉痛等等,因此導致了在受感染初期無發明確分辨是何種病毒所造成的窘境。實驗室先前的研究中發現到CD133癌幹細胞非常容易受到登革病毒的感染。因此我們假設CD133+神經幹細胞在受到登革與茲卡病毒感染時,可能會產生不同的感染狀態,並有機會能夠藉此不同之處區分出兩種病毒的感染。為進行此研究,我們選用了膠質母細胞瘤細胞株U87及神經母細胞瘤細胞株SK-N-SH兩種富含CD133癌幹細胞的神經癌細胞。我們發現U87及SK-N-SH兩種細胞都能分別受到登革病毒及茲卡病毒的感染。有趣的是,受到登革病毒感染的CD133細胞群能夠持續不斷地產出具有感染力的登革病毒;相反地,受到茲卡病毒感染的CD133族群則會隨著時間慢慢死亡,產生的感染曲線也逐漸降低。經由流式細胞儀分析後,我們發現在SK-N-SH細胞株中的癌幹細胞族群具有高表現的CD44。CD44除了是一種代表型的癌幹細胞之外,普遍被認為是一種參與多種調控細胞功能反應的黏附型分子;其亦為玻尿酸的在細胞上的受體,並在皮膚幹細胞受到登革病毒感染時扮演著重要的角色。為進一部探討登革病毒與茲卡病毒在影響生物功能上的不同之處,我們將神經幹細胞分選出並使用與玻尿酸結合的方式進行阻斷CD44表現的實驗,觀察是否能夠藉此對兩種不同病毒的感染曲線造成影響。我們從實驗結果中發現玻尿酸似乎能夠輕微地降低登革病毒的感染,然而在茲卡病毒感染的組別中卻無此現象。此結果說明在神經細胞中,CD44癌幹細胞可能也扮演著登革病毒受體的角色。由於CD44具有多種亞型,我們將使用siRNA來抑制CD44的基因表達,以確立CD44對登革病毒感染之重要性。在經過siRNA誘導而使細胞的CD44基因表現受抑制後,我們再次進行登革病毒感染的實驗,而結果顯示在受到感染後,細胞中登革病毒的RNA表現的確有明顯降低。綜合以上,我們認為神經幹細胞在受到登革與茲卡病毒感染後會在生物功能表現產生不同。
英文摘要 Zika virus (ZIKV), an arbovirus which belongs to Flaviviridae Flavivirus family, is well-known to infect neural stem cells and progenitors of the developing central nervous system. Previous studies also show that dengue virus (DENV) can infect neural cells resulting in encephalitis. In addition to similarity in genetic materials, these two viruses are not only co-circulating in tropical and subtropical regions but also share many common clinical symptoms in infected patients, including rash, fever and arthralgia. As such, it remains a big challenge to distinguish the two viral infections apart from each other. Glioblastoma (U87) and Neuroblastoma (SH-N-SH) are neuronal cell lines which have major cancer stem cells (CSCs) marker, CD133. Previous data from our lab suggested that CD133+ stem cells were highly permissive to DENV infection. We therefore hypothesize that a differential permissiveness of CD133+ neural stem cells to DENV and ZIKV infections may exit in nature. In this study, we found that CD133+ CSCs in the two cell lines could be infected by both DENV and ZIKV since both infectious viruses could be recovered from the infected supernatants. Interestingly, the integrity of DENV-infected CD133+ populations in SK-N-SH was observed, which could constantly shed DENV. In contrast, ZIKV-infected CD133+ populations were dying over time resulting in the decreasing of viral titer. FACS analysis showed CSCs populations in SK-N-SH expressed CD44 molecule on its surface. CD44 is known to be a homing cell adhesion molecule (HCAM), and has been shown to participate in a wide variety of cell functions. CD44 is a receptor of hyaluronic acid (HA), and recently it has been shown to play an important role in DENV infection in skin stem cells. To further confirm the differential biological functions for the two viruses in neural stem cells, we then sorted out the neural stem cells and performed CD44 blocking assay with HA prior to viral infections. Results showed a reduction of DENV titers after the HA blocking, suggesting that CD44 cancer stem cells might be a potential receptor for DENV but not in ZIKV. Since there are multiple isoforms of CD44 expressing on neuronal stem cells, and to testify the essential role of CD44 for DENV infection, we took an action by knocking down CD44 gene through siRNA silence assay. Results suggested that DENV RNA was significantly reduced in the neuronal stem cells after siRNA knocking down the CD44 gene. In summary, the biological differences in neural stem cells for DENV and Zika viral infections could be further investigated.
論文目次 中文摘要...........................................................................................I
Abstract..........................................................................................III
Acknowledgment.............................................................................V
Table of contents...........................................................................VII
List of figures.................................................................................X
Abbreviation Index..........................................................................XI
Introduction.....................................................................................1
1. DENV epidemiology.....................................................................1
2. ZIKIV epidemiology.....................................................................1
3. Cancer stem cells hypothesis and evidences.............................2
4. Properties of CSCs.....................................................................3
5. CD133+ CSCs.............................................................................3
6. CD44+ CSCs...............................................................................4
7. Role of bone marrow and neural stem cells in DENV infection.....4
8. Hypothesis..................................................................................5

Materials & methods........................................................................6
A. Materials...................................................................................6
1. Cell lines and virus.......................................................................6
2. Antibodies...................................................................................6
3. Antibodies (isotype).....................................................................7
4. Magnetic beads............................................................................7
5. Medium and Reagent....................................................................7
6. Ingredients in buffer and medium.................................................8
7. Equipment....................................................................................10
8. Instruments and machines...........................................................11
B. Methods....................................................................................13
1. Cell culture (U87, SK-N-SH, BHK21 and Vero)..............................13
2. Dengue virus expansion...............................................................13
3. Zika virus expansion.....................................................................14
4. Dengue virus infection..................................................................14
5. Zika virus infection........................................................................15
6. Plaque assay (DENV and ZIKV).....................................................16
7. Magnetic beads isolation...............................................................16
8. Purity checking..............................................................................17
9. Multicolor FACS analysis...............................................................18
10. Tumor sphere formation...............................................................18
11. Hyaluronic acid (HA) blocking assay.............................................19
12. CD44 siRNA knockdown...............................................................19
13. Statistical analysis........................................................................19
Results.................................................................................................21
1. U87 cells and SK-N-SH cells were both permissive to DENV and ZIKV infections…........................................................................................................21
2. Prolonging infections of SK-N-SH whole cells by ZIKV were observed......21
3. Sorted CD133+ cells from SK-N-SH were permissive to both DENV and ZIKV but only DENV infection could prolong the infection……………………..………………22
4. Sorted out CD133+ cells infected by DENV could survive and continue shedding the virus.............................................................................................23
5. ZIKV induce higher apoptosis rate of CD133+ cells resulting in reduction of viral titer…..........................................................................................................23
6. DENV and ZIKV infections did cause notable effects on tumorgenicity of CD133+ cells......................................................................................................24
7. HA treatment to block CD44 expression on CD133+ sorted from SK-N-SH cells reduced DENV infection.............................................................................25
8. Knocking down CD44 gene in SK-N-SH cells by siRNA reduced DENV RNA in these cells..........................................................................................................26
9. Knocking down CD44 gene of SK-N-SH stem cells by siRNA also reduced DENV RNA in these cells....................................................................................26
Discussion..........................................................................................................28
References..........................................................................................................32
Figures................................................................................................................37


List of figures
Figure 1. U87 cells and SK-N-SH cells were both highly permissive to both DENV and ZIKV infections…………………………………………………………………………....37
Figure 2. Kinetics of viral titers were similar in neural cells after DENV and ZIKV infections. ………………………………………………………………………………....38
Figure 3. Sorted CD133+ cells from SK-N-SH were permissive to both DENV and ZIKV but only prolong the viral titer after DENV infection. . …………………………………..39
Figure 4. Portion of the sorted out CD133+ cells which infected by DENV may be remained and keep releasing virus. . …………………………………………………………………40
Figure 5. ZIKV induced higher apoptosis rate of CD133+ cells than dengue virus than DENV did. . . ………………………………………………………………….. …………………41
Figure 6. CD133+ cells which infected by DENV kept producing higher viral titer after coculture with new cells. . . ……………………………………………………………….42
Figure 7. DENV and ZIKV infections did cause notable effects on tumorgenicity of CD133+ cells. . . ……………………………………………………………….. . . ………………..43
Figure 8. CD44 expression level could be blocked after high level HA treatment and reduced DENV infection in CD133+ from SK-N-SH cells…...…………………………………….44
Figure 9. CD44 siRNA reduced the dengue NS1 gene expression in SK-N-SH cells…….45
Figure 10. CD44 siRNA also reduced the dengue NS1 gene expression in SK-N-SH stem cells………………………………………………………………………………………...46
參考文獻 1. Rathore, A. P. S., et al. (2019). "Dengue virus–elicited tryptase induces endothelial permeability and shock." The journal of clinical investigation 129(10): 4180-4193.
2. Screaton, G., et al. (2015). "New insights into the immunopathology and control of dengue virus infection." Nature reviews immunology 15(12): 745-759.
3. Deepak, M., et al. (2014). "Dengue encephalitis-A rare manifestation of dengue fever. " Asian pacific journal of tropical biomedicine 4(1):70-72.
4. Gubler, D. J. (1998). "Dengue and dengue hemorrhagic fever." Clinical microbiology reviews 11(3): 480–496.
5. Kyle, J. L., et al. (2008). "Global Spread and Persistence of Dengue." Annual review of microbiology 62(1): 71-92.
6. Swaminathan, S., et al. (2009). "Dengue: recent advances in biology and current status of translational research." Current molecular medicine 9(2): 152-173.
7. Ioos, S., et al. (2014). "Current Zika virus epidemiology and recent epidemics." Médecine et maladies infectieuses 44(7): 302-307.
8. Shao, Q., et al. (2017). "The African Zika virus MR-766 is more virulent and causes more severe brain damage than current Asian lineage and dengue virus." Development 144(22): 4114-4124.
9. Enfissi, A., et al. (2016). "Zika virus genome from the Americas." The Lancet 387(10015): 227-228.
10. Dasti, J. I. (2016). "Zika virus infections: An overview of current scenario." Asian Pacific Journal of Tropical Medicine 9(7): 621-625.
11. Song, B. H., et al. (2017). "Zika virus: History, epidemiology, transmission, and clinical presentation." Journal of neuroimmunology 308: 50-64.
12. Muñoz, L. S., et al. (2017). "Neurological Implications of Zika Virus Infection in Adults." The journal of infectious diseases 216: 897-905.
13. Singh, S. K., et al. (2004). "Identification of human brain tumour initiating cells." Nature 432(7015): 396-401.
14. Soltysova, A., et al. (2005). "Cancer stem cells." Neoplasma 52(6): 435-440.
15. Alison, M. R., et al. (2008). "Stem cells and cancer: a deadly mix." Cell and tissue research 331(1): 109-124.
16. Reya, T., et al. (2001). "Stem cells, cancer, and cancer stem cells." Nature 414(6859): 105-111.
17. Bahmad, H. F., et al. (2019). "Cancer Stem Cells in Neuroblastoma: Expanding the Therapeutic Frontier." Frontiers in molecular neuroscience 12: 131.
18. Wu, X. Z. (2008). "Origin of cancer stem cells: the role of self-renewal and differentiation." Annals of surgical oncology 15(2): 407-414.
19. Dawood, S., et al. (2014). "Cancer stem cells: implications for cancer therapy." Oncology 28(12): 1101-1107, 1110.
20. Beachy, P. A., et al. (2004). "Tissue repair and stem cell renewal in carcinogenesis." Nature 432(7015): 324-331.
21. Tarayrah, L. and X. Chen (2013). "Epigenetic regulation in adult stem cells and cancers." Cell and bioscience 3(1): 41.
22. Alison, M. R., et al. (2011). "Cancer stem cells: problems for therapy?" The journal of pathology 223(2): 147-161.
23. Glumac, P. M. and A. M. LeBeau (2018). "The role of CD133 in cancer: a concise review." Clinical and translational medicine 7(1): 18.
24. Shah, K. (2016). "Stem cell-based therapies for tumors in the brain: are we there yet?" Neuro-oncology 18(8): 1066-1078.
25. Cheng, J. X., et al. (2009). "How powerful is CD133 as a cancer stem cell marker in brain tumors?" Cancer treatment review 35(5): 403-408.
26. Li, Z. (2013). "CD133: a stem cell biomarker and beyond." Experimental hematology and oncology 2(1): 17.
27. Lan, X., et al. (2013). "CD133 silencing inhibits stemness properties and enhances chemoradiosensitivity in CD133-positive liver cancer stem cells." International journal of molecular medicine 31(2): 315-324.
28. Zhu, Z., et al. (2017). "Zika virus has oncolytic activity against glioblastoma stem cells." Journal of experimental medicine 214(10): 2843-2857.
29. Yin, S., et al. (2007). "CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity." International journal of cancer 120(7): 1444-1450.
30. Ma, S., et al. (2008). "CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway." Oncogene 27(12): 1749-1758.
31. Senbanjo, L. T., et al (2017). "CD44: A Multifunctional Cell Surface Adhesion Receptor Is a Regulator of Progression and Metastasis of Cancer Cells." Frontiers in cell and developmental biology 5: 18.
32. Goodison, S., et al. (1999). "CD44 cell adhesion molecules." Molecular pathology 52(4): 189-196.
33. Ouhtit, A., et al. (2018). "Novel CD44-downstream signaling pathways mediating breast tumor invasion." International journal of biological science 14(13): 1782-1790.
34. Gao, A. C., et al. (1997). "CD44 is a metastasis suppressor gene for prostatic cancer located on human chromosome 11p13." Cancer Research 57(5): 846-849.
35. Horak, C. E., et al. (2008). "The role of metastasis suppressor genes in metastatic dormancy." Journal of pathology, microbiology and immunology 116(7-8): 586-601
36. Naor, D., et al. (2002). "CD44 in Cancer." Critical reviews in clinical laboratory sciences 39(6): 527-579.
37. Deed, R., et al. (1997). "Early-response gene signalling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells. Inhibition by non-angiogenic, high-molecular-weight hyaluronan." International journal of cancer 71(2): 251-256.
38. Lopez, J. I., et al. (2005). "CD44 attenuates metastatic invasion during breast cancer progression." Cancer research 65(15): 6755-6763.
39. Calisher, C. H., et al. (1989). "Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera." Journal of general virology 70(1): 37-43.
40. Kalayanarooj, S. (2011). "Clinical Manifestations and Management of Dengue/DHF/DSS." Tropical medicine and health 39(4): 83-87.
41. Srikiatkhachorn, A. (2009). "Plasma leakage in dengue haemorrhagic fever." Thromb haemost 102(6): 1042-1049.
42. Basu, R., et al. (2016). "Zika Virus on a Spreading Spree: what we now know that was unknown in the 1950’s." Virology journal 13(1): 165.
43. Rather, I. A., et al. (2017). "Zika Virus Infection during Pregnancy and Congenital Abnormalities." Frontiers in microbiology 8: 581.
44. Simmons, C. P., et al. (2012). "Dengue." New England journal of medicine 366(15): 1423-1432.
45. Chen, Q., et al. (2018). "Treatment of Human Glioblastoma with a Live Attenuated Zika Virus Vaccine Candidate." mBio 9(5).
46. Merfeld, E., et al. (2017). "Potential mechanisms of Zika-linked microcephaly." Wiley interdisciplinary reviews developmental biology 6(4).
47. Perng, G. C. (2012). "Role of Bone Marrow in Pathogenesis of Viral Infections." Journal of bone marrow research 1.
48. Zöller, M. (2015). "CD44, Hyaluronan, the Hematopoietic Stem Cell, and Leukemia-Initiating Cells." Frontiers in immunology 6: 235.
49. Wang, L., et al. (2018). "The Role of CD44 and Cancer Stem Cells." Methods in molecular biology 1692: 31-42.
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