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Title: | 以C57BL/6老鼠研究B型肝炎病毒基因型B和C所引起之免疫反應模式 The pattern of immune response between HBV genotypes B and C in the C57BL/6 mouse model |
Authors: | Mou-Mei Wang 王慕梅 |
Advisor: | 陳培哲 |
Keyword: | B型肝炎病毒基因型,核抗原反轉現象,高壓注射法,核抗原啟動子突變,C57BL/6小鼠, HBV genotype,HBeAg seroconversion,Hydrodynamic injection,BCP mutant,C57BL/6 mice, |
Publication Year : | 2009 |
Degree: | 碩士 |
Abstract: | B型肝炎病毒基因型B和C是包括我們台灣在內,亞洲常見的兩個基因型。根據臨床上的觀察,感染B型肝炎病毒基因型C會併發較嚴重的肝病,並有高危險率發展成肝癌。除此之外,在慢性感染過程當中,B型肝炎病毒基因型C的病人會有較高的比率在病毒核及核心抗原的啟動子發生突變,以及較高的比率發生核抗原反轉的延遲。因此,這兩項因素被認為是造成肝細胞癌的主因。
為了要探討B型肝炎病毒基因型B和C造成如此不同的免疫反應,我們使用實驗室已建立的小鼠免疫耐受模式,此模式是選用C57BL/6 小鼠以高壓注射法(Hydrodynamic injection)將帶有B型肝炎病毒基因並具有複製能力的質體pAAV/HBV1.2從尾靜脈導入老鼠體內。根據研究的結果,我們發現在打入B型肝炎病毒基因型B或C的小鼠表現差不多量的表面抗原和核抗原。此外,這兩個抗原的清除率在打入基因型B或C的小鼠上也是差不多的。至於產上抗體的情形,除了基因複製體 (clone) B7 以外,打入其他基因複製體的老鼠有百分之五十以上在核抗原消失的一個月後都尚未產生核抗體,這暗示著B型肝炎病毒基因型B和C在老鼠身上都產生延遲核抗原反轉的情形。為了觀察另一個影響核抗原反轉的因素,我們建立了帶有核抗原啟動子雙點突變的基因複製體。同樣以高壓注射法打入原生型和突變型的複製體做比較,我們發現打入啟動子突變型複製體的小鼠會產生較低的核抗原。此外,只有突變型複製體又帶有B型肝炎病毒基因型C的序列會在老鼠身上發生核抗原反轉延遲現象。令我們感到有趣的是,在表面抗原方面,打入突變型複製在體誘發老鼠大量表現後,表面抗原隨之快速被清除。 總結上述的實驗結果,我們發現在已建立的小鼠耐受模式下,B型肝炎病毒基因型B和C誘導產生相似的免疫反應於C57BL/6小鼠體內。對於研究的主要觀察目標-核抗原的反轉情形,在B型肝炎病毒基因型B和C的小鼠內都看到相同延遲的現象,並且只有B型肝炎病毒基因型C的啟動子突變型才有觀察到如同原生型的延遲反轉現象。然而這些結果並不能驗證臨床上病人感染B型肝炎病毒基因型B和C所看到的現象。未來我們所要設法改善的是要延長B型肝炎病毒在小鼠中的表現,比照臨床慢性感染的情形;並且要避免相同基因型的複製體間所造成的變異,如此才可避免我們比較B型肝炎病毒基因型B和C之間造成的誤差。 HBV genotypes B and C are two common genotypes in Asian region, including Taiwan. According to clinical observation, genotype C infection is associated with more severe liver diseases and higher risks of hepatocellular carcinoma development. Besides, genotype C carriers have higher frequency of basal core promoter (BCP) mutation and delayed HBeAg seroconversion during chronic HBV infection. Therefore, it is suggested that the genotype C and BCP mutation are two main factors contributing to HCC development. In order to explore the different immune responses between HBV genotypes B and C, we used the established immunocompetent mouse model, in which the C57BL/6 mice were introduced with the replication-competent plasmid pAAV/HBV1.2 by hydrodynamic injection. We found that C57BL/6 mice express the similar levels of serum HBsAg and HBeAg after injection of HBV genotypes B and C. In addition, the clearance rate of serum HBsAg and HBeAg were also similar in mice injected by HBV genotypes B and C. As to antibody production, with the exception of clone B7, over 50% mice had not generated serum anti-HBe more than one month since the HBeAg was cleared, indicating the delayed HBeAg seroconversion occurred in both HBV genotypes B and C. To investigate the other factor for HBeAg seroconversion, we constructed the clone with basal core promoter (BCP) double mutation. After hydrodynamic injection, comparing with the wild type, the mice injected by BCP mutants showed lower level of serum HBeAg. Moreover, the delayed HBeAg seroconversion was only observed when the mice were injected by the mutant clone of HBV genotype C. Interestingly, serum HBsAg level, on the other hand, declined rapidly after boosting in mice injected by BCP mutants of both genotypes B and C. Taken together, the overall expressions of HBV were not different significantly between HBV genotypes B and C in the C57BL/6 mice model, indicating their similar immune response induced after hydrodynamic injection. Moreover, regard to HBeAg seroconversion, mice injected by HBV genotypes B or C showed delayed HBeAg seroconversion, whereas only the mice injected by BCP mutant of genotype C could reproduce the same phenomenon relevant to the wild type. Therefore, these data suggested that we could not address the clinical observation of HBV genotypes B and C in this developed mouse model. Further studies are needed to prolong the HBV persistent expression of genotypes B and C in the mice and avoid clone-dependent variation for comparison. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43910 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 微生物學科所 |
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