- 學位論文
探討在人類基底部細胞株中HPV 18型E6及E7扮演的角色
Study the role of HPV-18 E6 and E7 in a human BCC cell line
摘要
人類乳突病毒 (Human papillowmavirus, HPV)是一環型雙股DNA病毒，依感染後引發的疾病致死率不同而分類為高危險群及低危險群。所有高危險群感染形成惡性腫瘤比例中又以HPV-16、18型占多數，HPV病毒的E6及E7為主要致癌蛋白，HPV E6或E7嵌入至宿主DNA中調控p53及Rb而影響細胞的生長及凋亡。人類基底細胞癌 (Basal cell carcinoma, BCC) 屬於皮膚癌症之一，暴露過量UV射線、燒燙傷害是造成皮膚基底細胞癌的危險因子。經由病毒感染而造成皮膚基底癌的比例甚少，根據統計，所有HPV感染皮膚基底細胞比例約27.8%，其中高危險型態感染比例約占23.1% 。本篇利用由一位台灣女性臉部燒燙傷的皮膚組織所培養出的皮膚基底細胞癌細胞株：BCC-1/KMC進行探討。 我們利用西方墨點法發現在BCC-1/KMC細胞中具有HPV-18 E6及E7的感染，接著利用免疫螢光染色及免疫沉澱法證實在BCC-1/KMC細胞中HPV E6經由與p53作用而調控p53蛋白表現。之後我們利用inducible si-RNA system 抑制HPV E6作用細胞生長遲緩，同時偵測細胞週期標誌蛋白發現HPV E6可能是透過p53影響p21表現，進而調控細胞週期的進行。如果給予細胞DNA微量傷害，發現細胞可能走向凋亡路徑，對於抑制E6後，細胞走向衰老或凋亡的調控機制，仍待後續實驗證明。 另一方面，同樣也利用免疫螢光染色及免疫沉澱法證實E7經由與Rb作用，進而影響細胞週期。利用inducible si-RNA system 抑制HPV E7作用後發現細胞生長遲緩，之後利用SA-β-gal試驗觀察到細胞走向細胞衰老。HPV E7藉由Rb影響細胞衰老已被廣泛討論，除了Rb外E7是否可影響其他路徑來調控細胞衰老，之後可繼續深入探討。
並列摘要
Human papillowmaviruses (HPV) are circular double-stranded DNA viruses that are classified by their transforming properties: the high-risk and low-risk types. The high-risk types of HPV involved in carcinogenesis almost are HPV type 16 and 18. HPV’s E6 and E7 are oncogenes that intergrated into host cell and regulated cell growth and apoptosis by modulating p53 and Rb. Basal cell carcinoma (BCC) is one of the skin cancers that exposure UV radiation and trauma scald are risk factors of BCC. Although viral infection has low percentage in BCC, recently studies have shown HPV were detected in 27.9% of basal cell carcinoma. Among the HPV-infected BCC, 23.1% were high-risk types of HPV. The BCC-1/KMC cell line was derived from the facial BCC of a female patient on the thermal traumatic scar. In this study, we used BCC-1/KMC to demonstrate the role of HPV-18 infection on BCC cell. First, we used western blot to demonstrate HPV-18 E6 and E7 are present in BCC-1/KMC cell line. By immunoflourescence and immunoprecipitation, we showed HPV E6 interacted with p53 and E7 interacted with Rb proteins. To interfere E6 and E7, the inducible-siRNA systems were designed and the cells were selected for further investigation, The results showed when E6 was interfered, cell growth was arrested via p53、p21 activation. When the interfered cells were exposured to low dose of adriamycin (ADR), that led to apoptosis. We also used inducible-siRNA systems for HPV-18 E7, the results showed cell growth arrest and initiated senescence by interfering E7. Regulation of HPV E7 with Rb has been discussed frequently, whether there are other mechanisms for cell senescence without Rb is not clear. Therefore, we like to investigate these issues in near future.