Detection of Hepatitis B Virus X Gene Integration in Peripheral Blood Mononuclear Cell and Bone Marrow CD34+ Cells from HBV Related Liver Disease Recipients after Liver Transplantation_West China Medical Journal

Authors：

LAI Wei ,  LU Shichun

Department of Hepatobiliary Surgery and Liver Transplantation Center, Beijing You-an Hospital, Capital Medical University, Beijing 100069, P. R. China;

Corresponding author：

LU Shichun, Email: laiwei119@163.com

Keywords：

肝移植; 单个核细胞; 造血干细胞; 乙肝病毒; 整合

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【摘要】　目的　了解乙型肝炎病毒（HBV）X基因在HBV相关肝病肝移植受体术后外周单个核细胞（PBMC）和骨髓CD34+细胞内的整合情况及其对乙肝疫苗接种的影响。　方法　采集1999年6月-2005年11月25例HBV相关肝病肝移植受体的外周静脉血及其中23例的骨髓血，以密度梯度离心结合单克隆免疫磁珠分离法获取外周血单个核细胞及骨髓CD34+细胞后，提取细胞DNA。因HBV X基因的整合频率最高，设计HBV X基因的特异引物，进行HBV-Alu-PCR，终产物进行电泳并回收、连接载体、筛选扩增后测序，检测有无X基因整合。　结果　经PCR后电泳及测序分析，25例HBV相关肝病肝移植受体术后的PBMC内未检测出HBV X基因的整合，其中采集到骨髓标本的23例CD34+细胞中亦未检测到HBV X基因的整合。　结论　肝移植术后受体体内HBV微生态的剧烈改变，使HBV整合的基本条件丧失，在此情况下，外周免疫细胞及骨髓造血干/祖细胞不是发生HBV整合的适宜场所，乙肝疫苗接种效果与HBV X基因整合关系不明确。
【Abstract】　Objective　 To investigate whether hepatitis B virus HBV X gene integrates in peripheral blood mononuclear cell (PBMC) and bone marrow CD34+ cells from HBV related liver disease recipients after liver transplantation.　Methods　 Between June 1999 and November 2005, PBMC were obtained from 25 HBV related liver disease recipients after liver transplantation and bone marrow CD34+ cells obtained from 23 cases among them. The cellular DNA was extracted by DNA isolation and purification kit following the manufacture’s instructions. Specific primers to HBV X gene and to human Alu repeats were used to amplify the virus integration through a 3-round hemi-nest PCR. The PCR final product was judged by 1.2% agarose electrophoresis, ligated to T vector, proliferated in E. coil 5α and sequenced.　Results　 According to agarose electrophoresis and sequencing analysis, there were no HBV X gene integration in PBMC and bone marrow CD34+ cells from HBV related liver transplant recipients after surgery.　Conclusions　 Because of the radical change of HBV microecological environment in HBV related liver transplant recipients after operation, the fundamental condition of HBV integration has been lost, which led PBMC and bone marrow CD34+ cells not suit to HBV X gene integrate to human genome. And the impact of HBV X gene integration on HBV vaccination is still undefined.

Citation： LAI Wei,LU Shichun. Detection of Hepatitis B Virus X Gene Integration in Peripheral Blood Mononuclear Cell and Bone Marrow CD34+ Cells from HBV Related Liver Disease Recipients after Liver Transplantation. West China Medical Journal, 2011, 26(3): 351-354. doi: Copy

1.	Murakami Y, Minami M, Daimon Y, et al. Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen[J]. J Med Virol, 2004, 72(2): 203-214.
2.	Laure FD. Zagury AG. Saimot RC, et al. Hepatitis B virus DNA sequences in lymphoid cells from patients with AIDS and AIDS-related complex[J]. Science, 1985, 229(4713): 561-563.
3.	Wang Y, Lau HS, Sham JS, et al. Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellualr carcinogenesis[J]. Oncogene, 2004, 23(1): 142-148.
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6.	Block TM, Mehta AS, Fimmel CJ, et al. Molecular viral oncology of hepatocellular carcinoma[J]. Oncogene, 2003, 22(33): 5093-5107.
7.	Jayshree RS, Sridhar H, Devi GM. Surface, core, and X genes of hepatitis B virus in hepatocellular carcinoma: an in situ hybridization study[J]. Cancer, 2003, 99(1): 63-67.
8.	赖威, 卢实春, 李幼平, 等. 肝移植术后外周血单个核细胞与CD34+细胞中HBV DNA含量及复制状态检测[J]. 中华肝脏病杂志, 2007, 15(11): 852-853.
9.	金圣杰, 卢实春, 赖威, 等. 乙型肝炎相关性肝病患者肝移植后重建乙型肝炎主动面的初步研究[J]. 中华肝脏病杂志, 2008, 16(4): 261-264.
10.	Minami M, Poussin K, Brechot C, et al. A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome[J]. Genomics, 1995, 29(2): 403-408.
11.	高海峰, 屠虹. 肝癌中乙型肝炎病毒整合位点的分离鉴定[J]. 中华肝脏病杂志, 2004, 12(9): 567-568.
12.	Block TM, Mehta AS, Fimmel CJ, et al. Molecular viral oncology of hepatocellular carcinoma[J]. Oncogene, 2003, 22(33): 5093-5107.
13.	Murakami S. Hepatitis B virus X protein: a multifunctional viral regulator[J]. J Gastroenterol, 2001, 36(10): 651-660.
14.	Yasui H, Hino O, Ohtake K, et al. Clonal growth of hepatitis B virus-integrated hepatocytes in cirrhotic liver nodules[J]. Cancer Res, 1992, 52(24): 6810-6814.
15.	Zimonjic DB, Keck CL, Thorgeirsson SS, et al. Novel recurrent genetic imbalances in human hepatocellular carcinoma cell lines identified by comparative genomic hybridization[J]. Hepatol, 1999, 29(4): 1208-1214.
16.	Murakami Y, Minami M, Daimon Y, et al. Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen[J]. J Med Virol, 2004, 72(2): 203- 214.
17.	Laskus T, Radkowski M, Wang LF, et al. Detection and sequence analysis of hepatitis B virus integration in peripheral blood mononuclear cells[J]. J Virol, 1999, 73(2): 1235-1238.
18.	Wang HP, Rogler CE. Topoisomerase I-mediated integration of hepadnavirus DNA in vitro[J]. J Virol, 1991, 65(5): 2381-2392.
19.	Yuwen H, Hsia CC, Nakashima Y, et al. Binding of wild-type p53 by topoisomerase II and overexpression of topoisomerase II in human hepatocellular carcinoma[J]. Biochem Biophys Res Commun, 1997, 234(1): 194-197.
20.	Menne S, Tennant BC. Unraveling hepatitis B virus infection of mice and men (and woodchucks and ducks)[J]. Nat Med, 1999, 5(10): 1125- 1126.
21.	Hwong CL, Chen CY, Shang HF, et al. Increased synthesis and degradation of DNA topoisomerase I during the initial phase of human T lymphocyte proliferation[J]. J Biol Chem, 1993, 268(25): 18982-18986.

1. 　Murakami Y, Minami M, Daimon Y, et al. Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen[J]. J Med Virol, 2004, 72(2): 203-214.
2. 　Laure FD. Zagury AG. Saimot RC, et al. Hepatitis B virus DNA sequences in lymphoid cells from patients with AIDS and AIDS-related complex[J]. Science, 1985, 229(4713): 561-563.
3. 　Wang Y, Lau HS, Sham JS, et al. Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellualr carcinogenesis[J]. Oncogene, 2004, 23(1): 142-148.
4. 　Buendia MA. Hepatitis B viruses and hepatocellular carcinoma[J]. Adv Cancer Res, 1992, 59: 167-226.
5. 　Robinson WS. Molecular events in the pathogenesis of hepadnavirus- associated hepatocellular carcinoma[J]. Annu Rev Med, 1994, 45: 297-323.
6. 　Block TM, Mehta AS, Fimmel CJ, et al. Molecular viral oncology of hepatocellular carcinoma[J]. Oncogene, 2003, 22(33): 5093-5107.
7. 　Jayshree RS, Sridhar H, Devi GM. Surface, core, and X genes of hepatitis B virus in hepatocellular carcinoma: an in situ hybridization study[J]. Cancer, 2003, 99(1): 63-67.
8. 　赖威, 卢实春, 李幼平, 等. 肝移植术后外周血单个核细胞与CD34+细胞中HBV DNA含量及复制状态检测[J]. 中华肝脏病杂志, 2007, 15(11): 852-853.
9. 　金圣杰, 卢实春, 赖威, 等. 乙型肝炎相关性肝病患者肝移植后重建乙型肝炎主动面的初步研究[J]. 中华肝脏病杂志, 2008, 16(4): 261-264.
10. 　Minami M, Poussin K, Brechot C, et al. A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome[J]. Genomics, 1995, 29(2): 403-408.
11. 　高海峰, 屠虹. 肝癌中乙型肝炎病毒整合位点的分离鉴定[J]. 中华肝脏病杂志, 2004, 12(9): 567-568.
12. 　Block TM, Mehta AS, Fimmel CJ, et al. Molecular viral oncology of hepatocellular carcinoma[J]. Oncogene, 2003, 22(33): 5093-5107.
13. 　Murakami S. Hepatitis B virus X protein: a multifunctional viral regulator[J]. J Gastroenterol, 2001, 36(10): 651-660.
14. 　Yasui H, Hino O, Ohtake K, et al. Clonal growth of hepatitis B virus-integrated hepatocytes in cirrhotic liver nodules[J]. Cancer Res, 1992, 52(24): 6810-6814.
15. 　Zimonjic DB, Keck CL, Thorgeirsson SS, et al. Novel recurrent genetic imbalances in human hepatocellular carcinoma cell lines identified by comparative genomic hybridization[J]. Hepatol, 1999, 29(4): 1208-1214.
16. 　Murakami Y, Minami M, Daimon Y, et al. Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen[J]. J Med Virol, 2004, 72(2): 203- 214.
17. 　Laskus T, Radkowski M, Wang LF, et al. Detection and sequence analysis of hepatitis B virus integration in peripheral blood mononuclear cells[J]. J Virol, 1999, 73(2): 1235-1238.
18. 　Wang HP, Rogler CE. Topoisomerase I-mediated integration of hepadnavirus DNA in vitro[J]. J Virol, 1991, 65(5): 2381-2392.
19. 　Yuwen H, Hsia CC, Nakashima Y, et al. Binding of wild-type p53 by topoisomerase II and overexpression of topoisomerase II in human hepatocellular carcinoma[J]. Biochem Biophys Res Commun, 1997, 234(1): 194-197.
20. 　Menne S, Tennant BC. Unraveling hepatitis B virus infection of mice and men (and woodchucks and ducks)[J]. Nat Med, 1999, 5(10): 1125- 1126.
21. 　Hwong CL, Chen CY, Shang HF, et al. Increased synthesis and degradation of DNA topoisomerase I during the initial phase of human T lymphocyte proliferation[J]. J Biol Chem, 1993, 268(25): 18982-18986.

West China Medical Journal

Detection of Hepatitis B Virus X Gene Integration in Peripheral Blood Mononuclear Cell and Bone Marrow CD34+ Cells from HBV Related Liver Disease Recipients after Liver Transplantation

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