臺灣博碩士論文加值系統

中 文 摘 要
背景:
許多研究證實：感染人類乳突病毒 (Human papillomaviruses, HPVs)是造成子宮頸癌的一個相當重要的危險因子。但是在口腔癌的發生上，人類乳突病毒所扮演的角色還並不清楚，主要是因為研究文獻中，缺乏同時有正常組織作為對照比較的資料，以及人類乳突病毒的重要性可能被其他造成口腔癌的口腔危險因子如抽菸、喝酒和嚼檳榔的影響所覆蓋。
方法:
為了探討人類乳突病毒在口腔癌致癌機轉上所扮演的角色，本實驗利用來自103個口腔癌標本及30個正常口腔黏膜純化出的去氧核糖核酸 (DNA)，利用兩組位在人類乳突病毒中一致性較高的L1部份的引子對 (MY09/GP6+ 和 GP5+/GP6+)，進行兩次聚合酶鏈反應 (polymerase chain reaction, PCR)來增加人類乳突病毒的DNA量，以供偵測。同時配合人類乳突病毒生物晶片 (genechip) 來作人類乳突病毒定型 (typing) 的工作，以此方法可在一次反應中偵測出39種不同人類乳突病毒的基因型。在分析有口腔習慣者與無口腔習慣者兩組的人類乳突病毒盛行率之外，本實驗也探討p53蛋白的免疫組織化學染色 (immunohistochemical staining) 的表現與臨床病理參數間的關係，及利用biotinyl-tyramide-based 的原位雜交方法 (biotinyl-tyramide-based in situ hybridization) 來偵測人類乳突病毒的DNA是否真的位於口腔癌腫瘤細胞核內，以此方法可同時分辨出人類乳突病毒的存在方式是游離基因形式 (episomal form)，或是較易使細胞變性 (transformation) 的整合形式 (integrated form)。
結果:
本實驗發現整體而言，人類乳突病毒的感染率在口腔癌標本(51/103, 49.5%) 中顯著高於正常口腔黏膜標本 (6/30, 20%, P<0.01)；如將口腔癌患者分為有口腔習慣者與無口腔習慣者兩組後，無口腔習慣口腔癌患者的人類乳突病毒感染率 (31/51, 60.8%) 顯著高於有口腔習慣的口腔癌患者 (20/52, 38.5%, P<0.05)。另外，高危險基因型的人類乳突病毒和人類乳突病毒基因型 18在口腔癌標本的感染率 (分別是41.7%及 26.2%,) 也顯著高於正常口腔黏膜標本者 (分別是16.7%, P<0.05 及 0%, P<0.01)；無口腔習慣口腔癌患者的高危險基因型人類乳突病毒和人類乳突病毒基因型 18的感染率 (分別是54.9% 及 39.2%) 也顯著高於有口腔習慣口腔癌患者的感染率(分別是28.8%, P<0.001 及13.5%, P<0.05)。經統計分析後，感染高危險基因型人類乳突病毒及任何基因型人類乳突病毒的患者將比一般人有3.97及3.92倍的機會得到口腔癌。而p53蛋白的過度表現在人類乳突病毒感染的患者 (33.3%) 也明顯高於無人類乳突病毒感染的患者 (13.5%, P<0.05)。利用biotinyl-tyramide-based 的原位雜交方法，我們發現人類乳突病毒的DNA以小點 (punctate) 的方式出現在口腔癌腫瘤細胞之細胞核內。這表示人類乳突病毒DNA的存在方式是較易使細胞變性的整合形式。
結論:
本實驗認為人類乳突病毒，尤其是高危險基因型的人類乳突病毒可能在臺灣口腔癌，特別是無口腔習慣患者的口腔癌，的致癌機轉中扮演重要的角色。而人類乳突病毒基因型 18在臺灣口腔癌的致癌機轉中扮演的角色，似乎是比人類乳突病毒基因型 16來得重要。但是，其在口腔癌的致癌機轉中扮演的確切角色，尚須進一步探討。本研究中，認為無口腔習慣的口腔癌有別於有口腔習慣的口腔癌，是一種特殊的臨床疾病類別，它有明顯不同的臨床表現，病理特徵，及分子致癌機制。在臨床上，這類癌症好發於女性患者，好發位置在舌頭，最初表現為舌部潰瘍，並且患者常無任何口腔習慣。所有低度分化 (poorly-differentiated) 的鱗狀細胞癌皆屬於此類癌症。 與人類乳突病毒有較高的關聯性以及p53的過度表現，也代表這類癌症有著不同的病因與致癌機轉。

Abstract
Background:
Human papillomaviruses (HPV) infection is a significant risk factor for uterine cervical carcinomas. However, the role of HPV infection in the development of oral squamous cell carcinoma (SCC) is still unclear due to the lack of normal control samples for comparison and the masking effect of other risk factors such as areca quid, tobacco and alcohol on the etiologic role of HPV.
Methods:
In order to evaluate the etiologic role of HPV in oral carcinogenesis, DNA samples were purified from 103 oral SCC specimens and 30 normal oral mucosal specimens. A nested-polymerase chain reaction (PCR) was used to amplify the consensus L1 region of the HPV genome in DNA samples using two sets of HPV primers (MY09/GP6+ and GP5+/GP6+). This procedure combined with a genechip HPV typing was able to detect a broad spectrum of HPV types in our samples. The HPV prevalence rates in normal oral mocosa (NOM) samples, all SCC samples, oral habits (OH)-associated SCC samples, and non-OH-associated SCC samples were determined and compared between groups. The expression of p53 in oral SCCs was studied by immunohistochemistry and its correlation with clinicopathological parameters was analyzed by chi-square test. Furthermore, we used biotinyl-tyramide-based in situ hybridization (BTBISH) to localize the HPV DNA in the nuclei of SCC cells. This method was able to differentiate whether the HPV DNA was integrated into the host cell genome or only in an episomal form.
Results:
We found that the overall HPV-positive rate was significantly higher in oral SCC samples (51/103, 49.5%) than in NOM samples (6/30, 20%, P<0.01) and significantly higher in non-OH-associated SCC samples (31/51, 60.8%) than in OH-associated SCC samples (20/52, 38.5%, P<0.05). The positive rate of high-risk HPV types or of HPV type 18 was also significantly greater in all SCC samples (41.7% or 26.2%, respectively) than in NOM samples (16.7%, P<0.05 or 0%, P<0.01, respectively) and significantly greater in non-OH-associated SCC samples (54.9% or 39.2%, respectively) than in OH-associated SCC samples (28.8%, P<0.001 or 13.5%, P<0.05, respectively). High-risk HPV types and all HPV types had an Odds ratio of 3.97 (95% CI, 1.40-11.28, P=0.0097) and of 3.92 (95% CI, 1.48-10.39, P=0.006), respectively. The overexpression rate of p53 protein was significant higher in HPV-positive oral SCCs (33.3%) than in HPV-negative oral SCCs (13.5%, P<0.05). By BTBISH, we found multiple punctate signals in the nuclei of oral SCC cells. This finding suggests that the HPV DNA has been integrated into the genomes of some of oral SCC cells.
Conclusion:
Our results suggest that HPVs, particularly high-risk HPV types, may play an important role in the development of oral SCCs, especially the non-OH-associated oral SCCs. HPV type 18 seems to play a more important role than HPV type 16 in oral carcinogenesis in Taiwan. However, further studies are needed to elucidate the exact role of HPV type 18 in the development of oral SCC. Non-OH-associated oral SCC is considered to be a specific disease entity with distinct molecular, pathologic, and clinical characteristics. Clinically, this kind of cancer is more common in females than in males, located at the tongue, initiated with tongue ulcers, and not associated with any OH. All poorly-differentiaed SCCs belong to this group. High-degree HPV association and p53 overexpression also implicate the different etiologies and pathogeneses of this group of oral cancers.

目 錄
中文摘要……………………………………………………………………6
Abstract……………………………………………………………………8
Introduction………………………………………………………………10
Background…………………………………………………………………12
Purpose of this study……………………………………………………13
Literature Review
Part 1: Introduction of oral squamous cell carcinoma…………14
(i) Epidemiology
(ii) Epidemiology in Taiwan
(iii) Clinical presentation
(iv) Histologic features
(v) Grading of squamous cell carcinoma
(vi) Differenital diagnosis
Part 2: Introduction of Human Papillomaviruses…………………17
(i) General view of HPV
(ii) Pathogenesis of human papillomaviruses
(iii) The relation of oral squamous cell carcinoma to HPV
Part 3: Methods for detection and typing of HPVs………………24
(i) Compare the methods for detection: in situ hybridization, PCR, nested-PCR assay sensitivity
(ii) Compare the methods for typing: in situ hybridization, RFLP, sequencing, microarray
Part 4: Squamous cell carcinoma of young adults………………27
Part 5: p53………………………………………………………………29
(i). p53 general view
(ii) p53: Structure-function relationship
(iii). p53 protein function
(iv). p53 in tumors with emphasis on cervical and anal cancer which harbor HPV
(v). p53 in SCC of head and neck
 Materials and methods
Part 1: Patients and specimens………………………………………36
Part 2: DNA extraction…………………………………………………37
Part 3: HPV detection by nested-PCR…………………………………37
Part 4: HPV genotyping by DNA sequencing analysis………………38
Part 5: HPV genotyping by genechip…………………………………38
Part 6: Immunohistochemiscal staining for p53 protein…………39
Part 7: Biotinyl-tyramide-based in situ hybridization of HPV…40
Part 8: Statistical analysis……………………………………………41
 Result
Part 1: DNA extraction……………………………………………………42
Part 2: HPV genotyping……………………………………………………42
Part 3: Correlation between HPV positivity and clinicopathological parameters…………………………………………43
Part 4: Association of oral habits with clinicopathological parameters……………………………………………………………………43
Part 5: Odds ratio of all HPVs and high-risk HPV types…………43
Part 6: Immunohistochemical staining for p53 protein……………44
Part 7: Biotinyl-tyramide-based in situ hybridization of HPV…45
Discussion……………………………………………………………………46
Conclusion……………………………………………………………………52
Reference……………………………………………………………………53
Tables…………………………………………………………………………65
Figures………………………………………………………………………74

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