- 學位論文
探討杜莎藻萃取物抗第一型單純疱疹病毒之機制
Mechanism of Dunaliella algae extract against Type I Herpes Simplex Virus (HSV-1)
摘要
隱形眼鏡為目前常用之近視矯正方式之一，故其配戴過程之清潔不完全所引起的細菌及病毒感染亦日益增加，進而引發感染性角膜炎。與其他疾病相比之下較不常見，但若沒有及時治療，其將造成眼睛與視力無法回復。單純疱疹病毒性角膜(Herpes Simplex Virus type Ⅰ；HSV-1)則是一常見眼疾，嚴重者將造成視力受損。試驗中使用之杜莎藻(Dunaliella salina)萃取物含有大量的類胡蘿蔔素(carotenoid)，研究也發現其具如抗氧化、抗癌等功能。故本研究以HSV-1病毒感染兔角膜上皮細胞傷害，並探討杜莎藻萃取物是否對角膜上皮細胞具保護功效與其抗病毒之機制。試驗一以不同劑量之HSV-1病毒(10 MOI、1MOI、0.1MOI)作用於兔角膜上皮細胞24小時後以乳酸脫氫酶測定(Lactic dehydrogenase assay；LDH)評估病毒劑量對細胞之毒殺性。試驗二則以不同濃度之杜莎藻萃取物預先添加於SIRC細胞1.5小時後，再經劑量1 MOI之HSV-1進行病毒感染24小時，收取其上清液及細胞，以蛋白質電泳(SDS-PAGE)與西方墨點法(western blotting)檢測蛋白質表現量、細胞存活率測定(Cell Counting Kit-8；CCK-8)及病毒斑試驗 (Plaque assay)之變化。結果發現，預先添加杜莎藻萃取物再經HSV-1感染後之細胞與為添加之SIRC細胞在外觀型態上可觀察到有減少感染現象產生，至於蛋白質表現方面則發現SIRC細胞受到HSV-1病毒感染可誘導發炎相關訊息傳遞路徑的活化與降低IL-6 pathway (JAK2、STAT1、STAT3)的表現，進而調控HSV-1所誘導的炎症反應。藉此試驗探討杜莎藻萃取物是否可以合併已知的抗病毒藥物使用，以達到最佳抗病毒效果。
並列摘要
Wearing contact lens is a popular way for myopia correction. Incomplete cleaning processes cause the higher risk for bacteria and virus infection.. Herpes simplex keratitis is a common ocular disease, delay cure would make eye and vision permanent damage. Dunaliella salina extract is made of carotenoid. It has the antioxidant and anticancer effects. Therefore, this study investigated the mechanism of SIRC cells infected by HSV-1 and examined the antiviral capabilities of Dunaliella salina. In experiment 1, SIRC cells infected with 10, 1, 0.1 M.O.I HSV-1 for 24 hours and examined cell cytotoxicity by Lactic dehydrogenase (LDH) assay. In experiment 2, SIRC cells were pretreated with Dunaliella salina extract for 1.5 hours then infected with 1 M.O.1 HSV-1 for 24 hours. Culture supernatants and cell extracts were collected for Cell Counting Kit-8 (CCK), plaque assay, Enzyme-linked immunosorbent assay (ELISA) and western blotting. The results indicated that Dunaliella salina extract could decrease HSV-1 infection in SIRC cells and inhibits IL-6 pathway (JAK-2, STAT-1, STAT-3) to regulate the inflammatory response. The results showed the antiviral capabilities and the protective effects of Dunaliella salina prevents damage. According to our results, Dunaliella salina extract is a potential medicine for inhibiting the effects of type I herpes simplex virus on herpes simplex keratitis.