2011-05-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647706摘要：慢性B型肝炎病毒的感染，是造成肝癌的重要危險因子。對於慢型B型肝炎感染之患者，除了包括年紀，性別，肝硬化等的宿主因素之外，還有幾項病毒本身的因子，被證明和肝癌的發生有關。除了血清中的高病毒量之外，包含病毒基因型為C型，有基礎核心促進子(basal core promoter)的突變，以及大表面蛋白缺失(pre-S deletion)等病毒因子，也都被證實和肝癌的發生有關。同時出現兩個或更多的病毒因子，也被報導具有協同作用(synergistic effect)，產生更強的致癌力。儘管已經有一些實證來說明這些病毒因子的重要性，但仍然還有一些未解的謎題，更待吾人去釐清。首先，儘管每一個單獨的病毒因子，都和肝癌的產生有關，但結合數個因子之後的協同作用，仍有待更清楚的證明。其二，假設合併兩個或更多的危險因子，的確增加病毒的致癌力。我們想要瞭解的是，是否這些和肝癌相關的病毒變異，都發生在同一株病毒上。若答案是肯定的，那麼吾人可以假設單一隻病毒，具有更多肝癌相關的病毒變異，則致癌的能力越高。反之，若是這些病毒變異都是分佈在不同的病毒株上，則可以推論，這些具有不同變異的病毒，可能在肝癌產生的過程上，在不同的時期，具有不同的作用。 其三，在過去的研究中指出，每一個病人身上的病毒，其實是一群極為類似，但卻不盡相同的病毒株，稱之為準種(quasispecies)。這群準種的組成以及其複雜度，都已經被證實，會影響到慢性B型肝炎患者之病程進展。而吾人希望藉由複製-讀序的方法 (cloning-sequencing)，將B型肝炎病毒準種的角色，予以進一步的釐清。我們因此假設，單一B型肝炎病毒病毒株，若是具備所有和肝癌發生有關的變異，包括病毒基因型C型，基礎核心促進子(basal core promoter)的突變，以及大表面蛋白缺失，則會造成最大肝癌發生的危險。而替代的假設則為和肝癌相關之準種和肝癌無關之準種，具有明顯的差異。這些和肝癌相關之基因變異，在之前的研究都是分開在不同的基因體區段內，作直接定序取得結果，因此看到的是一個總和的結果，而非單隻的病毒，無法用來研究準種;另外由於蒐集將表現型清楚區分的大型研究族群有相當的困難，再加上不易取得保存完善之血清及肝臟組織，讓這樣的研究不易進行。在這個研究中，吾人將致力於(1)蒐集一個大而完善的病人族群，清楚的臨床資料，能將病人明確的區分至疾病不同的時期。同時也將取得病人的血清以及肝臟的組織; (2)釐清是否單一病毒株具有兩個或是兩個以上的肝癌相關之病毒變異，能夠比其他沒有或僅具有一個肝癌相關之變異的病毒株，具有更強的致癌力; (3)釐清是否和肝癌有關的準種不同於和肝癌無關之準種; (4)探討是否單一病毒株擁有所有和肝癌相關之基因變異，其在複製上擁有較佳的能力。在經由分析這項研究中大量的B型肝炎病毒序列之資訊後，我們預期能夠瞭解這些肝癌相關之病毒因子，致癌之協同作用及其機制。我們長期的目標，是期望能夠確認出另一項，除了血清中B型肝炎病毒量之外的重要致癌之病毒因子。在臨床上，對於預測慢性B型肝炎患者之預後，能有極大的助益，同時在生物學上，對於B型肝炎病毒相關之致癌機轉，也能有進一步的了解。<br> Abstract: Chronic infection with hepatitis B virus (HBV) is an important risk factor for the development of hepatocellular carcinoma (HCC). Currently, several host factors including age, sex and liver cirrhosis, as well as viral factors have been documented to be related with HCC development. Regarding to the viral factors, apart from the serum viral load, several viral genomic background, such as HBV genotype C, the presence of basal core promoter (BCP) mutation, and the presence of pre-S deletion have been reported to be associated with the development of HCC. The coexistence of two or more viral factors is also reported to cause higher risk of HCC development. Although these lines of evidence revealed that each viral factor is related to the progress of the HBV-related liver diseases, several critical issues remain to be clarified.First, although each viral factor is associated to the progress of the HBV-related liver diseases, the synergistic effect of two or more viral factors on the hepatocarcinogenesis is not well defined. Second if the presence of two or more viral factors can synergistically enhance the process of hepatocarcinogenesis, then it is interesting to clarify whether the viral variants occur in the same HBV genome or not. If yes, then it can be assumed that the more HCC-associated viral mutations occurs on the same viral strain, the higher hepatocarcinogenic potential of the viral strain is. On the other hand, if the changes of these HCC-associated viral mutations did not occur on the same viral strain, maybe different viral mutants carrying different HCC-associated mutations act synergistically or additively on the development of HCC in a different way. Third, prior studies suggested that the HBV population is composed of different viral strains with minor differences, which is termed as quasispecies, in patients with chronic HBV infection. The composition or the diversity of the HBV quasispecies has been documented to influence the clinical course of HBV carriers. With the method of cloning-sequencing, the role of the HBV quasispecies will also be analyzed.We thus hypothesize that single HBV viral strain harboring all the HCC-associated genomic characters, including genotype C, BCP mutation, and pre-S deletion carry the highest risk of HCC development. Alternatively, we hypothesize that the HCC-related viral quasispecies differ from that of HCC-unrelated quasispecies. These HCC-related HBV genomic variants were mainly identified by using direct sequencing of HBV subgenomic regions separately in previous studies. Besides, the lack of a large cohort of patients with clear clinical phenotypes and well-preserved serum/liver sample bank may make such studies difficult to conduct.In this proposal, we thus aim to (1) collect a large cohort of patients with different stages of HBV-related liver diseases and with available serum and liver specimens for analysis; (2) clarify whether the presence of the single viral strain harboring two or more HCC-related HBV mutations is associated with a higher risk of HCC than the viral strain with no or only one HCC-related HBV mutation; (3) clarify whether HCC-related viral quasispecies differ from that of HCC-unrelated viral quasispecies; (4) to investigate whether the HBV viralstrain harboring more HCC-associated mutation process a better functional fitness.With the analysis of the massive HBV genomic data obtained from the project, we wish to understand more about the mechanism of the synergistic effect of these HCC-related viral genomic factors. The long-term object is to identify another determining risk factor of HCC in addition to the serum HBV-DNA level. Clinically, it will be beneficial to predict the prognosis of HBV carriers more accurately. Biologically, a better comprehension of HBV–related carcinogenesis will be accomplished.