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研究生:蔡宜璇
研究生(外文):Yi-Hsuan Tsai
論文名稱:以C57BL/6小鼠建立表現干安能抗藥性B型肝炎病毒之轉殖基因動物模式
論文名稱(外文):Establishment of Lamivudine-resistant HBV HBV Hepatitis B Virus Transgenic animal model in C57BL/6 Mice
指導教授:吳肇卿
指導教授(外文):Jaw-Ching Wu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:64
中文關鍵詞:B型肝炎病毒肝安能抗藥性基因轉殖鼠C57BL/6小鼠
外文關鍵詞:HBVLamivudine-resistantTransgenic MiceC57BL/6 Mice
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Currently there are three hundred and fifty million chronic hepatitis B (CHB) patients around the world. CHB is also the most common cause of liver cirrhosis and hepatocellular carcinoma. After marketing in 1999, Lamivudine became the most commonly used drug in the treatment of CHB. However, the cumulative incidence of resistance after using lamivudine for five years is 70%. Amino acid mutations in polymerase gene of hepatitis B virus (HBV) can result in cross-resistance to different nucleos(t)ide analogues. Therefore, it is important to develop new drugs for those patients with lamivudine drug-resistance. C57BL/6 mice have high genetic stability and easy to keep, and are thus suitable for establish an HBV transgenic animal model. We set up the transgenic mice model by pronuclear microinjection with HBV.
This study was designed to establish an animal model platform for the evaluation of drug efficacy and potential toxicity in long-term use. It is known that when the virus possesses rtL180M or rtM204V dual mutations will become resistant to lamivudine. In this study, we extracted HBV DNA from chronic hepatitis B patient's serum before (M1840) or after (P3358) the occurrence of lamivudine resistance to construct wild type (without lamivudine resistance) and mutant (with lamivudine resistance) HBV genome.To analyze the expression of hepatitis B virus in transgenic mice, we measured HBV viral loads and hepatitis B surface antigen (HBsAg) levels in serum, and analyzed HBV DNA and HBV transcripts using Southern blot and Northern blot hybridization, respectively. HBsAg and hepatitis B core antigen (HBcAg) expression in liver tissue were shown by immunohistochemical staining.
In Northern blotting, 3.5kb, 2.4kb and 2.1kb HBV RNA transcripts mimicking transcription of HBV in human infection were observed. HBV replication with replication intermediates in transgenic mouse liver were demonstrated by Southern blot analysis. HBsAg and HBcAg were shown in transgenic mouse liver tissue sections by IHC staining. Interestingly, mild steatosis and inflammation were found in some tissue sections. After lamivudine treatment via tube feeding for 2 weeks, the average serum viral load of lamivudine sensitive transgenic mice reduced from 2.91x107 to 4.33x106, with a ratio of residual HBV of 14.43%. And the average serum viral load of lamivudine resistance transgenic mice reduced from 1.04x107 to 5.34x106, with a ratio of residual HBV of 53.58%.
In summary, we have successfully established transgenic HBV mice of both wild type and lamivudine-resistant HBV These animal models will be useful for further evaluation of potential new anti-HBV drugs discovered from screening using HBV stable-expression cell line and the potential toxicity in different tissues after long-term antiviral use which could not be observed in detail using cell line study.

背景:目前全球約有三億五千萬人為慢性B型肝炎帶原患者,該疾病同時也是最常造成肝硬化以及原發性肝癌的元兇之一,干安能於1999年上市後成為最常用以治療慢性B型肝炎的藥物,然而干安能所引發的病毒抗藥性第1年為24 %,在五年後會高達70%,且干安能所引發的胺基酸突變會形成病毒對其他藥物之交叉抗藥性,因此研發新藥,特別是對干安能抗藥性患者具有療效之新藥十分重要,建立可評估新藥效能之動物模式平台亦相當重要。C57BL/6小鼠是基因穩定性高且容易掌控,是適合研究實驗動物之一,不過HBV無法直接感染小鼠,因此使用利用顯微注射法建立轉殖基因小鼠動物模式。
實驗設計與方法:本實驗旨在建立可評估新藥效能之動物模式平台,已知當病毒具有rtL180M或rtM204V二種突變點時會對干安能產生抗藥性,本研究分別採用了具有干安能抗藥性病患抗藥性突變出現前後之血清中病毒基因作為骨架,建構成1.46倍,總長度4.7Kb之HBV DNA,C57BL/6品系小鼠為背景,建立不具抗藥性的野生型與具抗藥型之突變型基因轉殖小鼠,測量血清中病毒含量以及表面抗原,並且以南方墨點,北方墨點法以及肝臟切片染色等分析B型肝炎病毒在肝臟組織中的表現結果,之後持續給予兩個禮拜的干安能,觀察HBV病毒量的變化是否隨治療而有所差異。
結果:以南方墨點法、北方墨點法檢測證實轉殖基因能在小鼠體內穩定表現,且組織切片染色則可發現部分小鼠輕微之脂肪肝與肝臟發炎之現象。檢測小鼠用藥前後血清病毒量之結果,野生型之表現量為平均由2.91x107減至4.3x106,平均殘餘14.43%,突變型之表現量平均為由1.04x107減至5.34x106,平均殘餘53.58%,且肝臟之病毒RNA與免疫切片之結果皆有相同之趨勢,顯示野生型之HBV DNA基因轉殖鼠確實比突變型HBV DNA基因轉殖鼠對藥物反應敏感,而肝安能抗藥性基因轉殖鼠則對藥物販應相對較差。結論,我們已成功建立野生型與干安能抗藥性基因轉殖鼠,未來可利用此動物模式做為抗B肝病毒藥物篩選與評估長期使用可能的副作用的工具。


1. Abstract 1
2. 中文摘要 3
3. List of Abbreviations 5
4. Introduction. 6
Hepatitis B 7
Epidemiology 7
The structure and life cycle of hepatitis B virus 8
Natural history of chronic hepatitis B 10
Animal Models of HBV 11
Natural Animal Model of HBV 11
HBV-Infected Mammal Models 12
The Duck Hepatitis Virus Infection Model 12
The Woodchuck Hepatitis Virus Infection Model 13
Artificial Animal Model of HBV 13
Hydrodynamic-based in vivo transfection 13
Chimeric mice model 14
HBV Transgenic mice 14
Immunomodulatory drugs and antiviral agents 16
Antiviral resistance 17
5. Rationale, Hypothesis, Aims and Experimental Designs 19
Rationale and hypothesis: 19
Aims 20
Experimental Designs of this study 20
6. Materials and Methods 21
HBV DNA extraction from patient’s Serum 22
Construction of wild type and drug-resistant HBV plasmid 22
Ligation and transformation of polymerase chain reaction (PCR) products 23
Purification of Mini-plasmid DNA 24
Preparation of DNA for Pronuclear Microinjection 24
Establishment of HBV transgenic mice 25
Mouse serum sample preparation 25
Quantitative analysis of HBsAg 26
Quantitative analysis of HBV viral load 26
Genotyping of transgenic mice by PCR method 27
Tissue sampling of mice 27
Analysis of HBV RNA 28
Sample preparation 28
RNA Electrophoreses 28
Transfer RNA from gel to membrane 29
Pre-hybridization and Hybridization 29
Washing 30
Blocking and antibody solution 31
Detection 31
Histology and immunohistochemistry 31
Histology 31
Immunohistochemistry 32
Statistical analysis 32
7. Results 34
Construction of vectors 34
Screening of transgenic founder mice by PCR method 34
HBsAg in the serum 34
Confirmation of the HBV DNA expression in organs 35
HBV antigen expression 35
Lamivudine-treatment reduced the HBV replication. 35
HBV mRNA expression in the liver 36
8. Discussion 37
9. Conclusion 39
10. References 40
Figure 47
Figure 1. The strategy of 1.46x HBV DNA construction. 48
Figure 2. The map of plasmid Alb-2HS4. 49
Figure 3. Genotyping by PCR method. 50
Figure 4. Serum HBV-DNA levels in lamivudine-resistant HBV and lamivudine sensitive transgenic mice. 51
Figure 5. Serum HBsAg levels in male and female transgenic mice. 52
Figure 6. HBV DNA replicative intermediates in liver, kidney, cerebrum, intestine, testis, spleen, mesentery and cerebellum. 54
Figure 7. Immunohistochemical staining of HBcAg and HBsAg in liver tissues of lamivudine -resistant HBV (B00) and lamivudine sensitive transgenic (D00) mice. 55
Figure 8. Immunohistochemical expression of HBsAg and HBcAg in liver tissues of lamivudine sensitive transgenic mice after 2weeks of lamivudine administration via oral feeding tubes. 56
Figure 9. Immunohistochemical expression of HBsAg and HBcAg in liver tissues of lamivudine resistant transgenic mice after 2weeks of lamivudine administration via oral feeding tubes. 57
Figure 10 Viral suppression in lamivudine sensitive HBV transgenic mice line D14 and D23. 58
Figure 11. Viral suppression in lamivudine resistant mutant transgenic mice line B00 and B13. 60
Figure 12. Compare the reducing ratio lamivudine sensitve and lamivudine resistant of after lamivudine treatment. 62
Figure 13. HBsAg and HBcAg immunohistochemical staining of HBV transgenic mice kidneys. 63
Figure14. Intrahepatic HBV RNA replicative intermediates of HBV transgenic mice. 64

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