乙型肝炎相關肝病肝移植術后外周血單個核細胞和骨髓造血干/祖細胞乙型肝炎病毒X基因整合檢測_《華西醫學》

作者：

賴威 ,  盧實春

首都醫科大學附屬北京佑安醫院肝膽外科暨肝移植中心（北京，100069）;

關鍵詞：

肝移植單個核細胞造血干細胞乙肝病毒整合

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【摘要】　目的　了解乙型肝炎病毒（HBV）X基因在HBV相關肝病肝移植受體術后外周單個核細胞（PBMC）和骨髓CD34+細胞內的整合情況及其對乙肝疫苗接種的影響。　方法　采集1999年6月-2005年11月25例HBV相關肝病肝移植受體的外周靜脈血及其中23例的骨髓血，以密度梯度離心結合單克隆免疫磁珠分離法獲取外周血單個核細胞及骨髓CD34+細胞后，提取細胞DNA。因HBV X基因的整合頻率最高，設計HBV X基因的特異引物，進行HBV-Alu-PCR，終產物進行電泳并回收、連接載體、篩選擴增后測序，檢測有無X基因整合。　結果　經PCR后電泳及測序分析，25例HBV相關肝病肝移植受體術后的PBMC內未檢測出HBV X基因的整合，其中采集到骨髓標本的23例CD34+細胞中亦未檢測到HBV X基因的整合。　結論　肝移植術后受體體內HBV微生態的劇烈改變，使HBV整合的基本條件喪失，在此情況下，外周免疫細胞及骨髓造血干/祖細胞不是發生HBV整合的適宜場所，乙肝疫苗接種效果與HBV X基因整合關系不明確。
【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.

引用本文： 賴威,盧實春. 乙型肝炎相關肝病肝移植術后外周血單個核細胞和骨髓造血干/祖細胞乙型肝炎病毒X基因整合檢測. 華西醫學, 2011, 26(3): 351-354. doi: 復制

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.	賴威, 盧實春, 李幼平, 等. 肝移植術后外周血單個核細胞與CD34+細胞中HBV DNA含量及復制狀態檢測[J]. 中華肝臟病雜志, 2007, 15(11): 852-853.
9.	金圣杰, 盧實春, 賴威, 等. 乙型肝炎相關性肝病患者肝移植后重建乙型肝炎主動面的初步研究[J]. 中華肝臟病雜志, 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點的分離鑒定[J]. 中華肝臟病雜志, 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. 　賴威, 盧實春, 李幼平, 等. 肝移植術后外周血單個核細胞與CD34+細胞中HBV DNA含量及復制狀態檢測[J]. 中華肝臟病雜志, 2007, 15(11): 852-853.
9. 　金圣杰, 盧實春, 賴威, 等. 乙型肝炎相關性肝病患者肝移植后重建乙型肝炎主動面的初步研究[J]. 中華肝臟病雜志, 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點的分離鑒定[J]. 中華肝臟病雜志, 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.

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乙型肝炎相關肝病肝移植術后外周血單個核細胞和骨髓造血干/祖細胞乙型肝炎病毒X基因整合檢測

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《華西醫學》

乙型肝炎相關肝病肝移植術后外周血單個核細胞和骨髓造血干/祖細胞乙型肝炎病毒X基因整合檢測

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