生產B型肝炎病毒之細胞株(HepAD38)特性分析

Characterization of HBV-producing HepAD38 cell line

林珈卉(Chia-Hui Lin)

指導教授 ： 陳培哲

國立臺灣大學/醫學院/微生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601877

摘要

B 型肝炎病毒為急性肝炎(acute hepatitis)或慢性肝炎(chronic hepatitis)的主原 因之一，全世界約有 20 億人口感染過 B 型肝炎病毒，其中有多於 3 億 5 千萬人 為 B 肝表面抗原(HBsAg)陽性的慢性帶原者，而慢性肝炎有極高的比例演變成肝 硬化(cirrhosis)或是肝癌 hepatocellular carcinoma (HCC)，其死亡率高達 25%。重 要的是，B 型肝炎慢性患者均無法靠目前的藥物痊癒。B 型肝炎感染肝細胞有許 多機制目前都還不是很清楚，我們必需要建立一個穩定的 B 型肝炎感染系統，用 以研究這些未知的機制並找到慢性 B 肝可能的解藥。過去以來，B 型肝炎在細胞 模式上的感染一直都困難重重，要提升感染率就必須要額外添加 PEG 及 DMSO。 直到發現 HBV 受體 NTCP，這對細胞模式上的感染來說是相當大的進展。而最 近也發現除了 NTCP 以外應該還有其他宿主蛋白會幫助 HBV 進入細胞之後的進 程，使 HBV 可以成功感染肝細胞。因此我們希望可以朝著找到其他影響 HBV 感 染的宿主因子，首先第一步就是要建立一個穩定的細胞模式感染系統。而我們所 使用的是廣為人知的 HepAD38 細胞株，這是一個會自己產生 HBV 的細胞株。 我們以西方墨點法來分析 HepAD38 細胞內的蛋白，發現看不到任何表面蛋白的 訊號，為了找到原因，我們以西方、北方墨點法及細胞培養上的改變去分析 HepAD38 的表面蛋白。最後發現，在 HepAD38 誘導後繼代培養對於 HepAD38 表面抗原之表現有極大的影響，並且發現表面抗原的抗體 13H10 無法辨識到 D 基因型的 HBV。我們也試了幾支不同的抗體，找到了 E11E4 和 Bioss 是可以辨 識 D 基因型 HBV 的表面抗原。簡單來說，沒有繼代對於 HepAD38 表面蛋白的 產生來說是有幫助的，加上使用可以辨認 D 基因型表面蛋白的抗體，使之在西 方墨點法上可以測得 D 基因型表面蛋白的表現。

關鍵字

B型肝炎感染 ； 細胞株HepAD38

並列摘要

Among the 2 billion global populations once infected with Hepatitis B virus (HBV), 350 million people remain HBV positive carriers. These chronic Hepatitis B (CHB) patients have high risks of developing more severe forms of liver diseases, such as liver cirrhosis and hepatocellular carcinoma (HCC), with a mortality rate of 25%. More importantly, these HBV long-term carriers stay uncured, with relapses andreoccurrences, even under current anti-viral treatments. In order to find possible cure to HBV, establishing a stable HBV infection cell model system is required to investigate the many unknown HBV pathways. However, over the past few years, HBV infection has faced many limitations in cell culture. Although HBV infection can be achieved by adding the two essential components PEG and DMSO, the infection rate was greatly improved after the discovery of the HBV receptor, NTCP. Such discovery took a huge leap forward not only for HBV infection in cell culture, but also for other HBV-related studies. Recently, some research pointed out that other host factors, in addition to NCTP, may contribute to sequential viral pathways, specifically related to viral-host interactions, in both hepatocytes and culture cell lines after HBV entry. Therefore, we aim to examine other possible factors that can affect HBV infection. First of all, to establish a stable infection system in cell culture, HepAD38, a widely known cell line that can produce HBV after induction in tet-off system, was used to serve as the viral source for this study. To confirm that HepAD38 can synthesize functional infectious particles as indicated in previous literature, the protein extract from the cell line was examined using Western Blot, but the results showed no surface protein. A series of experiments were carried out to investigate why there was no surface protein in western blot. I hypothesize that the antibody affinity to recognize HBsAg epitopes, and the cell culture methods are possible factors to impact surface antigen protein detection. To investigate such matter, western blot, northern blot, and alternative ways to culture cells were used in this study. The results showed that the surface antigen antibody 13H10 was unable to recognize the epitope on HBV genotype D whereas other surface antigen antibodies, E11E4 and Bioss, yielded promising outcomes. Moreover, in cell culture, whether HepAD38 was passaged after induction has a huge influence on the expression of surface antigen. Passaging HepAD38 after induction could reduce surface protein quantity by almost ten folds in comparison to the ones without. This implies that the first Western Blot results showing no surface protein could be due to its inadequate protein amount collected. Overall, the results in this study suggest that only two surface antigen antibodies, E11E4 and Bioss, and cell culture methods determine whether the surface protein of genotype D would be recognized.

並列關鍵字

HepAD38 ； HBV ； HBV infection

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