B型肝炎擁有放鬆、環形且不完整雙股，大約為3200個核苷酸的DNA基因體，往往會造成急性、慢性肝炎、或肝硬化，甚至最終導致肝癌的產生。目前治療B型肝炎的藥物主要是利用核苷酸類似物，包括了Lamivudin, adefovir dipivoxil, 與最近所使用的entecavir，這些藥物皆能有效壓抑血清中的病毒含量，並且使肝發炎指標ALT數值降低，優化肝中的發炎及纖維化程度。在in vitro的實驗中，Lamivudine以及adefovir dipivoxil皆未能完全壓制肝細胞內cccDNA的形成，這也造成cccDNA被視為是可能造成用藥停止而疾病複發的原因，所以在定量肝細胞內的cccDNA就顯得格外重要。在此研究我們的目標(1)建立定量方法在福馬林固定的蠟塊肝細胞組織中，萃取並定量其中cccDNA，此檢體比起先前研究常用的冷凍組織更容易取得，並且保存也較方便。(2) 將建立好的方法，與冷凍檢體所測得的數值做比較，看是否有相關性。 在研究過程中，我們收集了同一病人包含了冷凍以及蠟塊組織檢體，並且將蠟塊檢體利用切片機收集10,um 20片的組織，經過Xylene去蠟萃取之後，我們成功的依照先前的研究方式定量出肝細胞內HBV全部的DNA，然而我們定量蠟塊組織中cccDNA是採用在先前研究方法的引子外多設計一對引字，形成巢式定量PCR方式，並且與先前研究所採用的DNase酵素反應做處理，不過，令人驚訝的是，經過DNase處理過在蠟塊中cccDNA會有明顯降低的情況，但是在冷凍檢體中的cccDNA並未有明顯的改變，所以此結果也顯示了經過DNase酵素前處理的步驟會影響最終定量的結果，因此，在定量蠟塊中cccDNA的含量時，僅利用一次PCR反應後，經純化後，再利用二次即時定量，在最終的結果也顯示了此方法與定量冷凍組織cccDNA的方法相關性甚高。在未來，利用抗病毒藥物做評估時，也可以利用此方法定量蠟塊組織中的HBV的基因含量。

Hepatitis B virus (HBV) is a small noncytopathic hepatotropic virus that causes acute and chronic hepatitis, liver cirrhosis and development of hepatocellular carcinoma. Treatment of HBV infected patients with nucleotide or nucleoside analogues including lamivudine, adefovir dipivoxil, and entecavir effectively inhibit serum viral load, induce normalization of serum transaminase level, and improve liver necroinflammation and fibrosis. However, evidence from in vitro studies revealed that covalently closed circular DNA (cccDNA) of HBV was considerably resistant to lamivudine treatment. In addition to lamivudine, adefovir dipivoxil could only partially but not completely inhibit HBV cccDNA formation. Unlike adefovir and lamivudine, entecavir was shown to be capable of reducing cccDNA levels in liver in several in vitro studies. Therefore, aims of this study are (1) to establish the method to quantify cccDNA in formalin-fixed, paraffin embedded (FFPE) liver tissues, which are more accessible than fresh frozen tissues; (2) to compare results of FFPE liver tissues with fresh frozen liver tissues. We collected paired liver tissue with FFPE liver tissues and fresh frozen liver tissue from chronic hepatitis B patients who had enrolled in clinical trials with antiviral treatment, such as lamivudine, adefovir dipivoxil, and entecavir. For each FFPE liver tissues, we collected samples consisting of 20 sections with 10μm in thickness. Following DNA extraction, we successfully quantified total HBV DNA levels by specific real-time PCR assays. Moreover, we quantified cccDNA in FFPE liver tissues by using quantitative nested PCR with newly designed outer primers. This new method can only specifically amplify cccDNA verified by enzyme treatment. In this step, we discovered that DNase may have enzymatic activity on cccDNA quantification process. Futhermore, we examined the necessity of the DNase treatment. The results demonstrated that DNase can’t influence the amount of cccDNA in fresh frozen tissue but can reduce great amount of cccDNA in paraffin embedded tissue. Therefore, this method without DNase treatment was able to specifically to amplify cccDNA in paraffin embedded tissue. In the future, this method will apply to investigate the dynamics of cccDNA level before and after different antiviral treatment.

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