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研究生:梁正權
研究生(外文):Jenq Chyuan Neo
論文名稱:探討B型肝炎病毒大型表面抗原的Pre-S1及Pre-S2突變在肝臟再生及癌化過程中所扮演的角色
論文名稱(外文):The Biological Role of Pre-S1 and Pre-S2 Mutations of the HBV Large Surface Antigen during Liver Regeneration and Carcinogenesis
指導教授:蔡亭芬
指導教授(外文):Ting-Fen Tsai
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:72
中文關鍵詞:B型肝炎病毒表面抗原肝臟再生
外文關鍵詞:HBVlarge surface antigenPre-Shepatocarcinogenesiscarcinogenesisliver regeneration
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Hepatocellular carcinoma (HCC) remains an important world-wide health issue, especially in Asia. The risk factors of HCC development include environmental and other nongenetic factors. Hepatitis B virus infection is the main leading cause of HCC. The roles of HBV in tumor formation may through direct and indirect mechanisms. The HBV proteins, such as HBx and Hepatitis B virus surface antigen (HBsAg) have been studied for decades to understand their mechanistic roles in hepatocarcinogenesis. The implementation of infant Hepatitis B vaccination has declined the prevalence of chronic Hepatitis B. However, some HBV mutants have occurred because of virus high mutation rate, antiviral therapy and immune pressures. The large surface antigen comprises Pre-S1, Pre-S2 and S region. Clinically, Pre-S mutation of HBV large surface antigen was found in the serum and HCC tissues of patients. To investigate the physiological role of large surface antigen with Pre-S1/S2 mutation during liver carcinogenesis and liver regeneration, Pre-S1/S2 mutant transgenic mice were generated with the sequences provided by collaborators in VGH-Taipei and NHRI. Pre-S1/S2 mutant/HBx double transgenics have also been generated to study the synergistic effects of these two HBV proteins during hepatocarcinogenesis. In the liver regeneration study, Pre∆S2 (VGH) transgenic showed M-phase delay after partial hepatectomy for 48 hours. Alanine aminotranferase level of the transgenic was still normal before the age of 12-month. However, significant ALT increase and HCC formation was found in the founder of Pre∆S2 (NHRI) A205. Synergistic effect of Pre-S mutant and HBx in carcinogenesis was showed, earlier ALT increase and HCC formation was found in double transgenics compared to the single transgenics. In our study, we proposed Pparalpha�� was upregulated in Pre-S mutant transgenics, and caused increased C-myc expression through the inhibition of microRNA Let-7C, finally promoted the hepatocarcinogenesis.
肝癌依然是全球重要的健康議題,特別是在亞洲。肝癌的危險因子除了基因本身,還有環境等其他非基因分子。B型肝炎病毒的感染是其中導致肝癌發生的重要因子。B型肝炎病毒可以透過直接或間接的機制參與在腫瘤的形成過程中。為了了解B型肝炎病毒的致病急轉,其病毒蛋白,包括HBx蛋白、表面抗原也被研究多年。雖然嬰兒時期注射疫苗的措施已經降低了B型肝炎病毒的感染率,但是因為病毒的高突變率,抗病毒治療,以及免疫壓力,一些B型肝炎病毒的突變種也因而出現。大型表面抗原(large surface antigen)涵蓋了Pre-S1、Pre-S2及S區域。臨床上,可以在病人的血清或肝癌組織中發現具有Pre-S區域突變的大型表面抗癌。為了研究具有Pre-S1或Pre-S突變的大型表面抗原在肝癌過程及肝臟再生中所扮演的生理角色,我們使用由台北榮總及國家衛生研究院合作者提供的Pre-S1、Pre-S2突變的大型面抗原的序列,建立了Pre-S突變大型表面抗原的轉殖基因小鼠。為了研究Pre-S突變大型表面抗原與HBx蛋白在肝癌過程中是否有加成作用,我們也建立了同時帶有這兩種基因的轉殖基因小鼠。在肝臟再生的研究中,當切肝48小時後,會發現在Pre-S2(榮總)突變轉基因小鼠中出現M phase延遲的現象。截至目前對丙胺酸轉胺酶(ALT)的檢測,在轉基因小鼠12個月大前依然維持在正常值。但是,在Pre∆S2 (國衛院) A205譜系原源種(founder)中,在21個月的時候發現了丙胺酸轉胺酶上升及肝癌產生的現象。Pre-S突變蛋白及HBx蛋白也被發現對肝癌的形成有加成作用,帶有兩種基因的小鼠比HBx轉基因小鼠更快出現丙胺酸轉胺酶上升及肝癌的產生。在我們的研究中提出了在Pre-S突變轉基因小鼠中,可能透過Pparalpha�悀W升,抑制了微型核醣核酸Let-7C,進而促進C-myc的上升並參與在肝癌產生的過程中。
Abstract i
摘要 iii
I. Introduction 1
I-1. Hepatocellular carcinoma (HCC) 1
I-2. Risk factors of hepatocellular carcinoma 1
I-3. Hepatitis B virus (HBV) 2
I-4. The roles of HBV in hepatocarcinogenesis 3
I-5. The occurrence of HBsAg mutants 6
I-6. Endoplasmic reticulum stress in Pre-S mutant-mediated hepatocarcinogenesis 7
I-7. Peroxisome-proliferator-activated receptor alpha (Ppara) and HCC 9
I-8. The roles of c-myc in liver cancer 10
I-9. The microRNA, Let-7C plays roles in liver cancer 10
I-10. The mechanistic studies of PPARa, Let-7C and c-myc in hepatocellular carcinoma 11
II. Materials and Methods 13
II-1. Plasmid construction 13
II-2. Transformation 13
II-3. Miniplasmid isolation 13
II-4. DNA fragment elution 13
II-5. Genomic DNA extraction 14
II-6. Polymerase chain reaction (PCR) 14
II-7. Southern blot analysis 15
II-8. Total RNA extraction 17
II-9. Reverse transcription 18
II-10. Slot blot 18
II-11. Real-time PCR 19
II-12. Mature miRNAs quantification 19
II-13. Tissue processing and paraffin-embedding 21
II-14. Hematoxylin and eosin (H&E) staining 21
II-15. Immunohistochemistry (IHC) 22
II-16. ALT measurement 23
II-17. Partial hepatectomy and liver regeneration 23
II-18. Statistical analysis 23
III.Results 24
III-1. Generation and characterization of Hepatitis B virus large surface antigen (HBsAg) mutants transgenic mice 24
III-2. The effect of Pre-S mutant protein during liver regeneration 26
III-3. The carcinogenic effect of Pre-S mutants in transgenics 28
III-4. The synergistic effect of Pre-S mutant and HBx protein in hepatocarcinogenesis 28
III-5. Molecular mechanism study of Pre-S mutant 30
IV. Discussion 32
IV-1. Generation and characterization of Hepatitis B virus large surface antigen (HBsAg) mutants transgenic mice 32
IV-2. The effect of Pre-S mutant protein during liver regeneration 33
IV-3. The carcinogenic effect of Pre-S mutants in transgenics 34
IV-4. The synergistic effect of Pre-S mutant and HBx protein in hepatocarcinogenesis 36
IV-5. Molecular mechanism study of Pre-S mutant 36
References 40
Figures 45
Appendix 72
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