劉俊人2006-07-262018-07-112006-07-262018-07-112003http://ntur.lib.ntu.edu.tw//handle/246246/23614慢性B 型肝炎急性發作的免疫致病機轉迄今尚未明瞭。病毒株變異或是宿 主的免疫反應改變導致病毒與宿主間的平衡狀態破壞，被認為是引發急性發作最 重要的因素，但何者為重仍有爭論。本研究欲以前瞻性研究及全長基因體分析來 探討病毒株變異與慢性B 型肝炎急性發作的相關性。縱使真正致病機轉仍未明， 臨床觀察及動物實驗常發現血清ALT 值升高前會出現病毒量驟升的現象，若能 解開此病毒量驟升之謎，應對B 型肝炎急性發作的致病機轉有進一步的了解。 病毒日產量極高，其複製脢又欠缺校正錯誤的能力，因此極有機會出現病毒變異 株，藉著逃脫宿主免疫能力攻擊或是具有較好的複製能力，使病毒量驟升，進而 引發B 型肝炎急性發作。之前許多相關研究並無法得到一致的結論，次基因体 的研究策略及欠缺適當的研究模式為主要因素；更重要的是其採樣進行病毒核酸 序列分析的時間點多在急性發作之中或後，因此無法推測這些病毒核酸變異是否 真正引發急性發作，或僅是代表宿主免疫攻擊後的產物。前瞻性研究及全長基因 體分析當可回答此一重要問題。 第一年計劃中，我們已更廣泛地收集14 位自發性或是在醫療介入後發生B 型肝炎急性發作的病患， 我們首先定期分析血清ALT 值和HBV DNA 以釐清血 清HBV DNA 和ALT 值升高的相對應關係。結果相當一致地，在13 位(93%)病 患ALT 值升高前都會有病毒量竄升的現象。為探討病毒發生變異是否引發急性 發作，我們特別在急性發作過程中的四個點收集血清並進行HBV 全長基因体分 析: 收錄病患時、血清病毒量增加到最高時、血清ALT 值達最高時、和急性發 作之後。每一病患在不同時間點得到的HBV 全長基因体分別與病患收錄時的 HBV 全長基因体比較並找出變異所在，之後我們與已知病毒免疫反應標的區氨 基酸序列相對比和利用體外短暫轉染法，來進一步探討這些變異可能代表的免疫 學或病毒生物學方面的意義。結果發現在病毒量竄升到最高點時，遑論是發生在 急性發作之前或是與急性發作同時發生，病毒基因體幾乎與病患收錄時的病毒基 因體保持一致。在急性發作過程中，體內出現不同主要病毒基因體的病患比例隨 發作進展而逐漸增高：在血清ALT 值升到最高值時其比例為36%；在急性發作 之後，其比例則增高為50%。基因體出現的變異絕大多數是多重核甘酸點變異或 次基因體缺損。我們的初步結果顯示急性發作與病毒發生變異無關。 就病毒長期演變的觀點來看，為探討急性發作後出現的病毒變異是否真會造 成新的急性發作，我們將收錄連續發生急性發作的病患及其系列血清，增殖定序 其HBV 全長基因体並加以比對，來釐清此一問題。倘若有出現變異株，為了解 這些變異株是否有比較好的複製潛能導致病毒量竄升，我們也將以體外短暫轉染 分析法比較病毒野生株和變異株的複製能力。最後，真正具致病性的是肝細胞內 的HBV ，而大部份研究僅探討血清中的病毒基因体。為釐清血清中的HBV 是否 能代表肝細胞內的HBV ，我們也將自相同病患在同一時間取得其肝及血清檢 體，進一步比較二者檢體中病毒基因体的異同。 第二年計劃中，我們將繼續完成上述工作。我們預期經由此前瞻性全長病毒 基因体研究，將能釐清病毒株發生變異是否為造成急性發作的重要因素，此一發現將對慢性B 型肝炎急性發作的免疫致病機轉有重要貢獻。The onset of acute exacerbation (AE) during the course of chronic hepatitis B is likely related to the break of balance between virus and host immune responses. Whether such a break of immune tolerance is triggered major by the changes of host immune status or by an alteration of HBV hepatitis B virus (HBV) genome still remains controversial. To address this issue, a prospective study from silent (asymptomatic) stage to acute exacerbation and a full-length sequencing strategy are needed. Although the underlying mechanism remains unknown, clinical observations and animal studies clearly show that an upsurge of viral load always precedes or sometimes coincides with AE in chronic hepatitis B. Thereby, a key to the understanding of these AEs seems to unravel the origin and identity of such a viral surge. To clarify these issues, in the first year, we have already collected the clinical and serological data from 14 patients who developed acute exacerbation of chronic hepatitis B spontaneously, after discontinuing lamivudine treatment, or during interferon treatment. We first regularly monitored the serum ALT levels and HBV DNA levels during the development of acute exacerbation. Our results consistently showed that serum viral load resurged before the maximal hepatitis activity in 13 (93%). We then performed full-length HBV genome sequencing from the serum samples obtained at 4 points: at enrollment, at the peak of serum viral load, at the peak of serum ALT level, and after acute exacerbation. We found that the viral genome at virologic peak remained almost identical to that at baseline in 12 (86%) of them. On the contrary, the viral genomes obtained after the development of hepatitis exacerbation are different from the corresponding one at baseline in 7 (50%). Our preliminary results suggested that the development of hepatitis B exacerbation was not related to the changes of HBV genome. Nevertheless, from a viral evolution point of view, we need to consider the origin of the HBV strains at baseline. Some viral strains may exist and remain inactive in the host for a long time before reactivation, as is the case of chemotherapy-induced AE. The other baseline viral strains, especially those from repeated spontaneous AE, may just represent survivors from previous episode of AE. These HBV survivors may either be new viral variants selected out from host immune surveillance in previous exacerbation, or they may remain the same strain as prior to preceding AE and again trigger the current episode of exacerbation. In the second year, the sequential serum dominant viral sequences in some individuals with several episodes of AE will be determined to clarify if the viral variant selected during first AE will trigger a new AE. The in vitro phenotype of the viral variants emerged during AE will be determined. The representation of the serum viral genome to the intra-hepatic one will be determined by comparing the dominant viral sequences in the serum and liver specimens obtained simultaneously from the same individuals. Finally, the serial serum cytokines such as TNF-α and IFN-γ will be determined to see if antiviral host factors become defective before AE. We expect providing evidence to clarify whether alteration of viral genome besides host factor is related to the trigger of AE. In longitudinal follow-up, we can further clarify the impact of viral variations on AE and the biologic behavior of the emerged viral variants.application/pdf27495 bytesapplication/pdfzh-TW國立臺灣大學醫學院內科B 型肝炎病毒急性發作全長基因体變異前瞻性Acute exacerbationhepatitis B virusvariantviral loadfull-lengthgenomeprospective[SDGs]SDG3reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/23614/1/912314B002402.pdf