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研究生:林孟璇
研究生(外文):Meng-Hsuan
論文名稱:人類基底細胞癌中HPV-18之E6/E7與IL-6/8表現之相關性
論文名稱(外文):The correlation of E6/E7 to IL-6/8 gene expression in HPV-18 infected basal cell carcinoma
指導教授:許國堂
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學分子毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:84
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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。


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.


目錄
頁次
壹、 中文摘要 1
貳、 英文摘要 2
叁、 縮寫表 4
肆、 緒論 6
一、皮膚基底細胞癌 6
二、人類乳突病毒Human papillomavirus (HPV) 8
1. 人類乳突病毒之發現與分類 8
2. 人類乳突病毒之結構組成 8
2.1上游調節區(upstream regulatory region ,URR) 9
2.2早期表現區域 (early regions): 9
2.3晚期表現區域 (late regions) 11
3. 人類乳突病毒之致癌機轉 12
3.1 HPV E6與抑癌基因p53 12
3.2 HPV E7與抑癌基因Retinoblastoma(Rb) 12
三、介白素-6/8 (Interleukin-6/8, IL-6/8) 14
1. 介白素-6 (Interleukin-6, IL-6) 14
1.1介白素-6的來源 14
1.2介白素-6的功能 14
1.3介白素-6在疾病和腫瘤中所扮演的角色 14
2. 介白素-8 (Interleukin-8, IL-8) 15
2.1介白素-8的來源 15
2.2介白素-8的功能 16
2.3介白素-8在疾病和腫瘤中所扮演的角色 16
四、HPV與cytokine之相關性 18
伍、 研究動機 19
陸、 材料與方法 20
1. 材料 20
1.1 酵素 20
1.2 kit 20
1.3 試藥 20
1.4 抗體 20
1.5 細胞株與質體 21
1.6 儀器 21
2. 實驗方法 22
一、 質體之構築 22
(1) 設計inducible HPV E6及E7序列 22
(2) 限制酶反應 (Restriction Enzyme digestion) 22
(3) psingle-tTS-shRNA質體接合作用(psingle-tTS-shRNA
ligation) 22
(4)轉型作用 (Transformation) 23
(5) Colony Polymerase Chain Reaction (Colony PCR) 23
(6)洋菜凝膠 (agarose gel)之電泳分析 23
(7)微量質體DNA抽取 (Miniprep) 24
(8)中量質體DNA抽取 (Midiprep) 24
(9)自動定序反應的製備 (autosequence) 25
二、 細胞培養與分盤 25
(1) 解凍細胞株 25
(2) 細胞株培養 26
(3) 細胞株分盤 26
(4) 細胞計數及培養 26
(5) 回凍細胞 27
三、 細胞轉染作用 (Transfection) 27
LipofectamineTM 2000 Transfection Reagent 27
四、 Stable clone的挑選及培養 28
(1) 經由G418篩選stable clone 28
(2) Stable clone的挑選 28
(3) Stable clone的培養 28
五、 蛋白質濃度定量分析 28
六、西方墨點法 (Western blot) 29
(1) SDS-聚丙烯醯胺板膠之製備與操作 29
(2) 抗體作用及偵測方法 29
七、細胞中 RNA萃取 30
八、cDNA的合成與鏈聚合酵素連鎖反應 31
(1)以DNaseⅠ處理細胞RNA 31
(2)RNA之反轉錄作用 31
(3)RT-PCR 32
九、細胞生長分析(MTT) 32
十、UVB的照射 33
柒、 實驗結果 34
一、 建立標的於HPV-18 E6 以及 HPV-18 E7 的inducible sh-RNA 系統模式 34
二、 將帶有抑制HPV-18 E6/E7表現的質體送入BCC-1/KMC 細胞內,並挑選穩定的細胞群落 34
三、 分析加入誘導劑Doxycycline後誘發sh-E6/ E7作用的效果 35
四、 分析加入誘導劑Doxycycline後誘發sh-E6對於其細胞內標的基因p53的互動 35
五、 分析加入誘導劑Doxycycline後誘發sh-E7對於其細胞內標的基因Retinoblastoma (Rb)的互動 36
六、 觀察在穩定表現sh-E6/E7的BCC-1/KMC細胞株中,抑制HPV E6/E7作用對於細胞生長的影響 36
七、 分析BCC-1/KMC細胞中HPV-18 E6/E7兩者間的關聯性 36
八、 探討UVB的損害對於感染HPV皮膚癌細胞所分泌IL-6以及IL-8的影響 37
九、 分析BCC-1/KMC細胞內抑制E6 / E7對於IL-6 mRNA 表現之影響 48
十、 分析BCC-1/KMC細胞內抑制E6 / E7對於IL-8 mRNA 表現之影響 38
捌、 討論 39
玖、 圖表及圖表說明 46
圖一、 利用半定量RT-PCR偵測在BCC-1/KMC細胞內抑制HPV E6及E7 mRNA之表現 47
圖二、利用西方墨點法偵測在BCC-1/KMC細胞內抑制HPV E6及E7及其相關之蛋白表現 49
圖三、利用西方墨點法分析在BCC-1/KMC細胞內抑制HPV E6作用及回復p53蛋白活性的效果 50
圖四、利用西方墨點法分析在BCC-1/KMC細胞內抑制E7作用,回復Rb蛋白活性的效果 51
圖五、利用MTT assay觀察在穩定表現sh-E6以及sh-E7的
BCC-1/KMC細胞株中,抑制HPV E6或E7作用對於細胞生長的影響 52
圖六、利用半定量RT-PCR以及西方墨點法分析在BCC-1/KMC細胞內抑制HPV E6或E7之後,對於兩者之間相互影響的關係 54
圖七、利用不同能量之UVB射線照射,分析HPV E6以及
E7相對於IL-6 以及 IL-8表現之相關性 56
圖八、 利用半定量RT-PCR偵測在BCC-1/KMC細胞內抑制HPV
E6或E7之後,對於IL-6 以及 IL-8表現之影響 58
壹拾、 附表及附圖 59
壹拾壹、 參考文獻 66


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