肝癌是目前全世界發生率高且具有致命性的癌症之一，雖然已經有多種不同的方式能夠應用於肝癌的治療，但是治療後高復發的情況仍然沒辦法被解決，由於癌幹細胞有生成腫瘤的能力並且對於某些化療藥物有抗藥性，在癌症治療之中根除癌幹細胞是有困難的，因此癌幹細胞與癌症的復發及轉移具有密切的相關性。先前研究指出登革病毒感染骨髓之中的造血幹細胞會影響細胞的分化，由於癌幹細胞具有和造血幹細胞相似的特性(例如:自我更新以及分化的功能)，再加上我們先前的實驗證實肝癌腫瘤組織中帶有CD133標誌的癌幹細胞確實能夠被登革病毒感染，因此本篇研究我們假設肝癌細胞株HepG2中的癌幹細胞應該也能被登革病毒感染，並且病毒的感染可能也會對這些癌幹細胞造成一些影響，主要著重於探討肝癌細胞株HepG2中帶有CD133和CD34標誌的肝癌幹細胞受到感染後細胞族群以及功能上是否產生變化，此外也想了解肝癌幹細胞受登革病毒感染後是否能繼續存活，讓病毒持續存在其中複製，利用癌幹細胞不斷產生具有感染力的新病毒。首先我們先確認肝癌細胞株HepG2能夠被登革病毒感染，並確定此細胞株中包含帶有CD133和CD34標誌的肝癌幹細胞，且帶有CD133標誌的肝癌幹細胞族群比例非常高，接著我們從HepG2細胞中分選出帶有CD133或CD34標誌的細胞並確認這兩種癌幹細胞皆能被登革病毒感染，我們也發現登革病毒的感染會造成癌幹細胞族群的變化，帶有CD133和CD34標誌的細胞受到感染之後比例會減少，且當帶有CD133標誌的細胞族群較少時，帶有CD34標誌的細胞可能會轉變成帶有CD133標誌的細胞，因此HepG2細胞中帶有CD133標誌的細胞族群比例高低與登革病毒感染後產生的病毒量沒有顯著的相關性，接著我們想了解癌幹細胞族群的變動是否和細胞凋亡有關係，因此在感染後不同時間點利用Annexin V染色觀察細胞凋亡的情形以及紀錄細胞存活的比例，結果顯示登革病毒感染並不會促進帶有CD133標誌的細胞產生細胞凋亡，且受感染後存活的細胞增加，然而帶有CD34標誌的細胞雖然有一部份會走向細胞凋亡，但是存活的部份我們推測可能可以轉變成帶有CD133標誌的細胞，此外我們也證實登革病毒感染之後存活的癌幹細胞能夠讓病毒在其中繼續複製，產生更多具有感染力的病毒，最後藉由電子顯微鏡的觀察我們發現登革病毒感染進入帶有CD133標誌的癌幹細胞時可能會被包裹到單層模的囊泡中，並且病毒顆粒可能透過出芽(budding)的方式離開受感染的細胞，產生型態大小不一的非典型登革病毒顆粒，另外我們也觀察到細胞的狀態在感染36小時後和感染12小時及24小時相比有恢復的情況，這個現象支持了我們先前的推測，登革病毒能夠存在在帶有CD133標誌的細胞中不斷的複製且不會促進細胞凋亡發生。綜合上述實驗，我們發現登革病毒能夠感染HepG2細胞中帶有CD133和CD34標誌的癌幹細胞，並且感染之後會造成幹細胞族群的改變，實驗結果證實帶有CD133標誌的細胞受到登革病毒感染之後能夠繼續存活，病毒也能持續存在細胞中複製，此外當登革病毒感染帶有CD133標誌的肝癌幹細胞時可能會被包裹到單層膜囊泡中，且新的病毒顆粒可能藉由出芽的方式釋放，我們也推測或許此種方式讓帶有CD133標誌的細胞受感染後得以繼續存活，因此我們認為肝癌之中帶有CD133標誌的細胞對於登革病毒的感染以及複製扮演重要的角色。

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers in the world. Although different therapies have already been used to treat HCC, the high recurrence rate remains a big challenge. Relapse and metastasis of HCC are intimately related to cancer stem cells (CSCs), which are tumorgenic and show resistance to chemotherapy. Previous studies have demonstrated that dengue virus (DENV) infection can affect the differentiation of hematopoietic progenitor cells (HPCs) in bone marrow (BM). CSCs possess similar biological properties of HPCs, such as self-renewal and differentiation. Furthermore, previous study revealed that CD133+ cells sorted out from HCC tumor tissue were highly permissive to DENV infection and yet the sustainability of CD133+ cells infected by DENV remains to be explored. CD133+ CSCs of HCC cell line (HepG2 cells) were utilized to investigate the characteristics of CD133+ cells after DENV infection. Stem cell populations in HepG2 cells were quantified by FACS. Biological functions, morphologies of cells and viral particles in sorted stem cells were performed by colony forming unit assay (CFU), tumor sphere formation assay, and transmission electron microscopy (TEM). Results confirmed that HepG2 cells were infectable by DENV, and contained high percentage of CD133+ population. Sorted CD133+ cells from HepG2 cells were permissive to DENV infection, so did sorted CD34+ cells. Interestingly, the properties of stem cells were altered after DENV infection; sustainable for DENV infection in sorted CD133+ or decreased number of cells in CD34+ cells, and DENV infected CD34+ cells were capable of transforming to CD133+ cells. As such, the levels of CD133+ cells in HepG2 cells were not proportionally correlated to viral titers in HepG2 cells. Annexin V staining for live cells revealed that DENV infected CD34+ cells resulted in apoptosis, while apoptosis was not observed in infected CD133+ cells, and the percentage of live CD133+ cells was increased after DENV infection. Moreover, sorted CD133+ cells from 2-day DENV infected cells could produce infectious DENV after re-culturing in fresh media. TEM investigations indicated that multiple DENV may be packaged inside a vesicle-like structure in infected CD133+ cells. Unconventional viral particles in various sizes were budded out from the infected CD133+ cells. Images also showed that the morphology of CD133+ stem cells recovered at 36 hours post infection, suggesting that DENV might be able to get along with CD133+ cells after infection. Cumulative data showed that CD133+ or CD34+ stem cells in HepG2 were permissive to DENV infection, and the infection may alter population of these stem cells. CD133+ cells could survive after DENV infection and support continuous viral replication. In infected CD133+ cells, DENV may be packaged inside monolayer vesicles and the viral particles might bud out from the cell membrane without killing the cells. In conclusion, CD133+ cells could not only sustain DENV infection but also be a reservoir for propagation of the virus, suggesting that these cells play an important role in supporting DENV replication in HCC.

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