- 學位論文
人類基底細胞癌中HPV-18之E6/E7與IL-6/8表現之相關性
The correlation of E6/E7 to IL-6/8 gene expression in HPV-18 infected basal cell carcinoma
摘要
基底細胞癌(Basal cell carcinoma, BCC)是最常見的皮膚癌之一，而其特性為容易局部侵犯，但不易轉移；其致病的原因主要為陽光的過度曝曬，而燒灼傷和接種疫苗的傷疤而引起的皮膚病變也可能造成。 在許多皮膚疾病或腫瘤的研究中發現，當角質細胞受到某些外來的刺激時，便會產生促發炎激素而引發皮膚的免疫機制。過去研究也指出感染人類乳突病毒 (Human papillomavirus, HPV)16/38型之角質細胞，經由UVB照射之後，相較於正常角質細胞，會產生高量的IL-6及IL-8；因此學者推論，HPV病毒的感染，與皮膚細胞產生發炎反應的過程是有相關性的。 人類乳突病毒可依其致癌能力，分為高危險型和低危險型；高危險型中以HPV-16、18形成惡性腫瘤的比例占多數。而致癌的過程主要是由於致癌蛋白E6及E7對於腫瘤抑制因子p53及Rb的調控所導致。本實驗利用由一位台灣女性臉部燙傷疤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。首先我們以西方墨點法證實BCC-1/KMC具有HPV-18 E6及E7的感染，接著利用inducible sh-RNA system將HPV-18 E6及E7做為目標進行干擾，加入誘導劑Doxycycline將E6/E7抑制之後，確實能使p53及Rb的蛋白表現恢復。同時， 我們也發現E6/E7兩者的調控機制是有相互關聯性的，而非獨立的作用。 另一方面，我們也想進一步探討在皮膚細胞在癌化的過程中，致癌蛋白E6/E7是否也對於細胞激素的產生扮演著重要的角色。因此我們先利用半定量RT-PCR，偵測當E6/E7受到抑制之後，對於IL-6及IL-8表現的調控。並且利用UVB射線模擬對於感染HPV皮膚癌細胞的損害，對於其所分泌細胞激素中IL-6以及IL-8的影響。在此我們初步提出認為具有HPV病毒感染的皮膚癌細胞，其致癌基因以及發炎激素，對於皮膚癌細胞惡化的過程中，是有顯著相關性的；而HPV-18 E6及E7是否對於其他發炎相關的激素具有調控或影響的能力，仍待後續實驗深入的探討。
關鍵字
皮膚基底細胞癌並列摘要
Basal cell carcinoma (BCC) is one of the most commonly skin cancer in humans and is characterized as locally aggressive slowly growing tumor with rarely metastatic potential. Skin exposed to sunlight is considered to be a major etiologic factor for the pathogenesis of BCC. In addition, either trauma scald or vaccine inoculation cause skin pathological change may increase the incidence. Keratinocytes can be induced to produce cytokines by several exogenous stimuli and dysregulation of this production has been described in various skin diseases, including cancer. The release of these cytokines can trigger a cutaneous inflammatory. It has been demonstrated previously that UVB irradiation strongly upregulates IL-6 and IL-8 secretion in human keratinocytes immortalized with HPV-16 or HPV-38 and suggest an active role of these viruses in modulation of the skin inflammatory process. Human papillomavirus (HPV) can be classified into high-risk or low-risk groups according to the propensity for malignant progression of the associated lesions which they cause. The high-risk types of HPV involved in carcinogenesis almost are HPV type 16 and 18. Lines of investigation indicate that the two oncoproteins E6 and E7 are known to inactivate the major tumor suppressors, p53 and retinoblastoma protein (pRB), respectively. In addition, it has conferred the tumourigenic properties of transformed cells by multiple mechanisms. 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. To inhibit E6 and E7, the inducible-shRNA systems were designed and the coordinate inactivation of E6 and E7 as the target gene, are provided by the system that responds to the presence of the inducer doxycycline (Dox). We have demonstrated that p53 and Rb were upregulated by silencing HPV E6 and HPV E7. Furthermore, our data suggest that the regulation of E6 and E7 is an interrelated one but not independent function. Next, we asked whether the HPV E6 and E7 play an important role to the production of cytokine during the tumourigenesis in BCC cells. We used semiquantitative RT-PCR to detect the altered expression of IL-6 and IL-8 by silencing HPV E6 and HPV E7. We also detected the effect of UVB irradiation on secretion of IL-6, IL-8 that involved in the inflammatory process by HPV-infected skin cancer cell. These results suggest that cytokines may synergistically cooperate in the development of skin lesions. Whether there are other cytokines involved in inflammation and the correlation to HPV E6/E7 is not clear. Therefore, we plan to investigate these issues in near future.