以高壓流體注射之小鼠模式探討B型肝炎病毒基因型A與D之生物學差異

The Biology between Hepatitis B Virus Genotype A and D in the Hydrodynamically Injected Mouse Model

江若熙(Jo-Hsi Chiang)

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(2017年)

https://doi.org/10.6342/NTU201704052

摘要

B型肝炎是由B型肝炎病毒感染所引發的肝發炎反應。儘管現行的預防性疫苗有效地降低了B型肝炎的發生率，但在1982年疫苗問世前已感染B型肝炎病毒的人口數量龐大；目前全球約有2.6億慢性B型肝炎帶原者，而每年約90萬人死於慢性B型肝炎病毒感染造成的併發症。B型肝炎病毒目前已知有十種基因型(基因型A至J)，各基因型除了具特定的地域性分佈外，每種基因型對抗病毒療法的免疫反應也都不盡相同。以四種最常見的B型肝炎基因型為例，基因型A分布全球，是最廣泛的一種B型肝炎病毒基因型；基因型B和C是亞洲最常見的B型肝炎病毒基因型；而基因型D則是基因型A之外在歐洲最常見的B型肝炎病毒基因型。由於特殊的地域性分布，這四種基因型的傳染途徑也因此而有些差異。亞洲最廣泛分佈的基因型B和C大多由母子垂直感染途徑傳播，因為大部分感染者年齡偏低，而慢性B型肝炎感染其中的要素即是年齡，故此，大部分亞洲地區的感染者在疫苗問世之前會轉變為慢性帶原者。相較於基因型B和C，基因型A與D的傳播途經大多是在成人間平行傳染，因此較不容易形成慢性帶原；即使如此，基因型A和D仍然存在著一些顯著的差異。在同個地區，同樣群體，藉由相同傳染途徑及感染年齡相仿的這些條件之下，感染基因A的群體卻比感染基因型D的群體容易轉變為慢性帶原者，但是在抗病毒藥物的治療下，基因型D的感染者對干擾素的反應卻遠不及基因型A。有鑑於此，本篇研究希望藉由高壓流體注射小鼠模式去探討基因型A與D這兩者在生物學上的異同以及可能的致病機轉，而本篇研究的假說為。實驗結果顯示，在高壓流體注射之後三天犧牲的C57BL/6小鼠肝臟樣本中，B型肝炎病毒基因型D核心蛋白的表現量顯著地高於基因型A，而這與血清中e抗原的表現量以及HuH-7細胞模式所得出的結果是相應的。相較於核心蛋白的表現量，在HuH-7細胞模式中，基因型A的表面抗原蛋白表現量是遠優於基因型D，這與兩個基因型在細胞培養液和小鼠血清中的表面抗原表現量相呼應。總括而言，B型肝炎病毒基因型A傾向表現較大量的表面抗原蛋白，而基因型D則較傾向表現較大量的核心抗原蛋白。雖然此二種特性與兩種基因型在臨床上的關聯性仍須更進一步的研究，現階段的發現能夠為後續兩基因型的研究提供一個有效的研究平台。

關鍵字

B型肝炎病毒基因型 ； B型肝炎病毒D基因型 ； B型肝炎病毒A基因型 ； 高壓流體注射小鼠模式 ； 表面抗原 ； 核心抗原

並列摘要

Hepatitis B virus (HBV) has long been a serious global public health threat. With 257 million worldwide chronic carriers, HBV prevails in a region-specific fashion, correlating to its genotypes as well as the means of transmission. Categorized by at least 8% genetic divergence from one another, ten HBV genotypes (A-J) have been identified to date, each with various subgenotypes. Among the four most common HBV genotypes (A-D), while genotype A is pandemic and genotype B and C rampage in Asia, genotype D is somewhat overlooked in spite of its predominance in the Mediterranean countries and the Middle East. Besides the most common transmission route difference (genotype B and C being vertical and genotype A and D horizontal), clinically genotype D displays genotype-specificity in both disease progression and treatment response. When comparing HBV genotype D with genotype A—both found in the same geographic region—patients with genotype D tend to show a weaker response to interferon treatment and a more severe acute liver disease such as fulminant hepatitis than those with genotype A. The well-established hydrodynamic-injection (HDI) technique was utilized here to create a transfection mouse model to investigate HBV genotype D against its genotype A counterpart. Judging by the clinical characteristics and previous scientific literature, I hypothesize that the HBV genotype D would display a lower tendency to persist in the HDI mouse model in comparison to its genotype A equivalent. The results showed that the core protein expression level of HBV genotype D in the HDI C57BL/6 mouse liver samples was about three times that of the genotype A samples. Such finding echoes the mouse serum titers of e-antigen (HBeAg) between the two genotypes as well as the HuH-7 cell model results. For surface protein expression, on the other hand, the HuH-7 cell model evidence suggested that HBV genotype A has about 25 times higher than that of genotype D in western blotting. However, the mouse serum samples and supernatant samples of culture HuH-7 cells only showed a two-to three-fold difference in HBsAg titer level between the two genotype (ELISA). This implies that such stark difference of surface protein expression in western blotting may be attributed to poor HBsAg antibody specificity that favors the recognition of HBV genotype A surface antigen over that of genotype D. Although more intensive studies are required to be carried out to draw meaningful connections linking the findings of this study to clinical significance of the two genotypes, the pAAV-HBV1.3 genotype D clone produced from this study can serve as a powerful tool for all future studies related to HBV genotype D in our laboratory.

並列關鍵字

HBV genotypes ； HBV genotype D ； HBV genotype A ； hydrodynamic injection mouse model ； core protein ； surface protein

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