以CBA小鼠模式探討中和肝細胞內外次病毒表面抗原顆粒對B型肝炎病毒持續存在之影響

Abstract

B型肝炎(Hepatitis B)是由B型肝炎病毒(Hepatitis B virus, HBV)所引起的一種具有潛在性危及生命的肝臟感染症。據世界衛生組織(World Health Organization, WHO)估計，全世界目前約有二億四千萬人罹患慢性B型肝炎，因此B型肝炎是一個不容忽視的全球性公共衛生議題。B型肝炎病毒轉譯出的表面抗原(HBsAg)計有大型(large, L)、中型(medium, M)、小型(small, S)等三種抗原型態。HBsAg除了會構成病毒表面的外套膜(envelope)之外，也會自我組裝形成不具感染性的次病毒顆粒(subviral particles)，因其不含HBV核酸之故。根據臨床研究發現，在感染HBV的病人血清中，次病毒顆粒產生的數量遠多於具感染性的B型肝炎病毒，約達1,000~10,000倍之多。目前廣為醫界所接受的學說，認為造成慢性B型肝炎感染的主要原因是感染者對HBV病毒所產生的免疫反應不足所導致，部分可能與病毒的免疫抑制功能有關。過去一些研究顯示，HBsAg抗原可能會抑制免疫細胞之功能。本實驗室建構了一種利用高壓注射法(hydrodynamic injection)將B型肝炎病毒核酸送入肝細胞的動物模式，使B型肝炎病毒得以長期持續在小鼠的肝臟細胞內表現，藉以模擬人類感染B型肝炎病毒後慢性帶原的現象。利用此一技術，我們希望藉由觀察小鼠對B型肝炎清除能力的改變，來探討B型肝炎病毒表面抗原的免疫調節功能。 在本研究中，我們利用B型肝炎病毒表面抗原的抗體，將肝臟細胞內以及肝臟細胞外的自由型表面抗原(free HBsAg)短暫地清除，再來偵測血清中的B型肝炎病毒e抗原(HBeAg)與麩丙酮酸轉胺脢(Alanine aminotransferase, ALT)的濃度有無改變。實驗結果顯示，利用抗體中和肝臟細胞外或肝臟細胞內的free HBsAg後，HBsAg抗原在血清中會急遽地下降，但大約4周後又會回復到和對照組相同的濃度，而HBeAg抗原也持續存在。本研究說明了利用抗體短暫地中和小鼠肝臟細胞內或外的自由型表面抗原並不會影響到小鼠對B型肝炎的清除。未來，希望利用各種不同HBsAg抗原的突變體找出其可能的免疫調節功能。

Hepatitis B is a potentially life-threatening persistent liver infection caused by the hepatitis B virus (HBV). According to the latest estimates of World Health Organization (WHO), more than 240 million people have chronic hepatitis B infection in the world. Therefore, it is a major global health problem. The HBV viral envelope consists of three related hepatitis B surface Antigens (HBsAg), the large (L), medium (M) and small (S) ones. In addition to the formation of viral envelope, the HBsAg can also be secreted as the non-infectious subviral particles, in which no viral genome exists. Based on clinical findings, the subviral particles are typically expressed 1,000~10,000 times higher than infectious virions in the sera of patients with HBV infection. Currently, it is widely accepted that chronic HBV infection is due to ineffective host antiviral immune response against HBV infection, which is thought to be partly caused by immune-suppressive viral molecules. Previous studies suggested that HBsAg, the viral envelope protein, could inhibit the function of immune cells. In the past, our laboratory has successfully established a technique called “hydrodynamic injection” to bring the nucleic acids of HBV into mouse hepatocytes, in which HBV proteins and viral particles can express and last for a long time, to mimic the human chronic HBsAg carriers. By using this technique, we hope to investigate the role of HBsAg in immune regulation through the observation of the clearance of HBsAg in mice. In this study, hydrodynamic injection of HBV DNA-expressing plasmid in CBA mice was conducted, and then anti-HBsAg antibody with two different concentrations was injected to block extracellular free HBsAg particles 24 hours later. In addition, HBV DNA plasmid along with anti-HBsAg antibody were hydrodynamically co-injected into hepatocytes. The levels of serum HBeAg and alanine aminotransferase(ALT) were determined after antibody injection. The results showed that the serum HBsAg levels were immediately depleted in both experiments. However, after four weeks the HBsAg levels bound back to control group level in both groups. The mice remained persistently seropositive for HBsAg for fourteen weeks in the extracellular group. The results suggested that a transient inhibition of extracellular free HBsAg particles and hydrodynamically co-injected with anti-HBsAg antibody along with HBV plasmid will not affect HBV persistence in CBA mice. In the future, we hope continue the study to understand more clearly the immune regulation mechanism in which the HBsAg involves by use of HBV mutants.