以非轉殖小鼠模式進行B型肝炎病毒感受性之基因體分析

Abstract

人類B型肝炎病毒感染後的結果與宿主的遺傳背景有關，儘管在同年齡感染B型肝炎病毒，有些人能夠將B型肝炎病毒清除，有些人則會形成慢性帶原者，但造成差異的調控機制與免疫反應，至今還不清楚。先前在高壓水動力注射的小鼠模式中發現，BALB/cJ小鼠能在注射後四周內快速的清除B型肝炎病毒，而同一時間內大部份的C57BL/6J小鼠則會持續性表現注入質體所包含的B型肝炎病毒。由於兩種小鼠飼養在相似的環境之中，所以我假設是兩種小鼠品系間的遺傳變異造成了不同的實驗結果。本研究目的即利用全基因表現量微陣列及自交品系小鼠所建構之遺傳圖譜兩種篩選方法，找尋造成實驗結果不同所參與的基因及調控機制。 在全基因表現量微陣列的結果中發現，BALB/cJ小鼠在注射B型肝炎病毒表現質體六小時與十八小時後，受第一型干擾素活化的基因表現量有顯著的上升。然而，第一型干擾素本身的改變則並不明顯。在適應性免疫相關基因的表現上，BALB/cJ小鼠在注射後第五天也有顯著地提高。適應性免疫的活化造成了BALB/cJ小鼠中B型肝炎病毒的清除與過去的研究結果相符，但第一型干擾素所參與的先天性免疫反應對與B型肝炎病毒感染所扮演的角色，在本研究中仍然無法清楚的證明，未來仍需要更多的實驗來釐清第一型干擾素的反應機制。 而在自交品系小鼠遺傳圖譜分析中，我們紀錄了11個自交品系在注射後八週的B型肝炎病毒清除趨勢，並利用這些小鼠所建構的遺傳圖譜找出與清除趨勢具高相關性的基因座。分析結果顯示在第8號染色體上92.98 Mb到94.57 Mb的位置與性狀分布有最高的相關性，此區段包含了Chd9、 Rbl2、 Aktip、 Rpgrip1l、 Fto及Irx3等六個基因。在各品系中B型肝炎病毒的表現性狀，以及定位圖譜的建構，都還有待進一步的實驗及分析增加定位的可信度。

The clinical outcomes after hepatitis B virus (HBV) exposure vary a lot among individuals. While some people rapidly clear the virus and generate effective antibodies against HBV, others fail to do so and become chronically infected. It is suggested that the differences among individuals may be contributed by their genetic variations. Nevertheless, the underlying genetic components, which may contribute to HBV clearance/persistence, are not known of. Using a hydrodynamic-based and immune competent mouse model, our group previously demonstrated that different inbred mouse strains have different response after HBV injection. The BLAB/cJ strain was able to eliminated HBV plasmid to an undetectable level two to three weeks after hydrodynamic injection, while 80% of C57BL/6J strain failed to clear the virus four weeks after injection. In order to systematically survey the underlying genetic components corresponding to HBV clearance in mouse, two strategies were applied, i.e. whole genome expression profile by microarray and in silico mapping. For expression microarray, I found that interferon-stimulated genes (ISGs) were significantly up-regulated in BALB/cJ at 6 and 18 hours after injection. In addition, expression levels of adaptive immune related genes were elevated in liver of BALB/cJ five days after injection. It has been proposed that the initiation of adaptive immunity against HBV is responsible for the viral clearance. The current study suggests that innate immunity may play an important role for HBV clearance. Nevertheless, there was only limited induction of type I interferon and was not significantly different between BALB/cJ and C57BL/6J. Therefore, how type I interferon involved in HBV infection is not clear at the moment and may need further investigation. For in silico mapping, a total of 11 inbred mouse strains were selected for analysis. The clearance patterns within eight weeks after HBV injection were served as the phenotypic trait and correlated to haploblocks constructed by CGD1 mouse SNP data set. One locus located at 92.98 Mb to 94.57Mb on chromosome 8 exhibits highest correlation score. This region contains six annotated genes, including Chd9, Rbl2, Aktip, Rpgrip1l, Fto, and Irx3. I found that the results of in silico mapping are strongly affected by phenotype defined, inbred strain used, and method of haploblock construction applied. In order to improve the results, one should try to use different phenotypes and more rigorous haploblock construction methods in further study.