一种转导人原代肝细胞的LV-HBx诱导调控表达系统

doi:10.3969/j.issn.1004-616x.2017.04.005

Carcinogenesis, Teratogenesis & Mutagenesis ›› 2017, Vol. 29 ›› Issue (4): 266-271.doi: 10.3969/j.issn.1004-616x.2017.04.005

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An inducible expression system of LV-HBx for transducing primary human hepatocyte

ZHOU Xiaoling^1,2,3, XIONG Xiaofang², XIE Qingdong^1,2,3, SUN Pingnan^1,2,3

1. Stem Cell P2 Laboratory, Shantou University Medical College, Shantou 515041;
2. Reproductive Medicine Center, Shantou University Medical College, Shantou 515041;
3. Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou 515041, Guangdong, China

Received:2017-04-06 Revised:2017-06-13 Online:2017-07-31 Published:2017-07-31

Abstract

Abstract:

OBJECTIVE: To construct an inducible lentiviral system which expressed HBx in primary human hepatocytes (PHH) for the study of biological characters of HBx and its influence on PHH. METHODS: We constructed a Tet-on inducible HBx-lentiviral expression system,and optimized it to a relatively high expression level. We applied this system to make LV-HBx and LV-rtTA recombinant lentivirus,to transduce HepG2 hepatoma cells and PHH,and to induce expression of HBx with doxycycline. RESULTS: We successfully constructed the recombinant lentiviral vector of HBx. The ratio of recombinant lentivirus of LV-HBx and LV-rtTA was optimized and a higher transduction efficiency could be approached at the ratio of 1:1 or 2:1 with HBx-positive cell ratio of 67.3% and 66.7%,respectively. This system was successfully applied in transducing HBx gene and inducing HBx expression with doxcycline in HepG2 and PHH. The expression level of HBx in PHH with Dox induction was about 45 fold of the group without Dox induction. HBx significantly increased the expression level of MTA1,VEGFA,and TGFB1 and decreased the expression level of CDH1 and IGFBP3,which is consistent with the previous reports. CONCLUSION: We successfully constructed a Tet-on expression system of LV-HBx for the transduction of HBx into PHH. This system will facilitate studies on influence of HBx and other HBV gene on PHH.

Key words: HBx, primary human hepatocyte, inducible expression, lentiviral vector

CLC Number:

Q291

ZHOU Xiaoling, XIONG Xiaofang, XIE Qingdong, SUN Pingnan. An inducible expression system of LV-HBx for transducing primary human hepatocyte[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2017, 29(4): 266-271.

References

[1] SYLVAN S. WHO spearheads global initiative to eradicate hepatitis B[J]. Lakartidningen,2000,97(35):3738-3740.
[2] TANG H,DELGERMAA L,HUANG F,et al. The transcriptional transactivation function of HBx protein is important for its augmentation role in hepatitis B virus replication[J]. J Virol,2005,79(9):5548-5556.
[3] LEUPIN O,BONTRON S,SCHAEFFER C,et al. Hepatitis B virus X protein stimulates viral genome replication via a DDB1-dependent pathway distinct from that leading to cell death[J]. J Virol,2005,79(7):4238-4245.
[4] HUANG J,KWONG J,SUN E C,et al. Proteasome complex as a potential cellular target of hepatitis B virus X protein[J]. J Virol,1996,70(8):5582-5591.
[5] TRUANT R,ANTUNOVIC J,GREENBLATT J,et al. Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation[J]. J Virol,1995,69(3):1851-1859.
[6] SLAGLE B L,ANDRISANI O M,BOUCHARD M J,et al. Technical standards for hepatitis B virus X protein (HBx) research[J]. Hepatology,2015,61(4):1416-1424.
[7] YANG S,SHI H,CHU X,et al. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors:useful for making iPS cells and transduction of primary cells[J]. Biotechnol Lett,2016, 38(9):1631-1641.
[8] SANDIG V,HOFMANN C,STEINERT S,et al. Gene transfer into hepatocytes and human liver tissue by baculovirus vectors[J]. Hum Gene Ther,1996,7(16):1937-1945.
[9] ZAMULE S M,STROM S C,OMIECINSKI C J. Preservation of hepatic phenotype in lentiviral-transduced primary human hepatocytes[J]. Chem Biol Interact,2008,173(3):179-186.
[10] LEE M H,NA H,NA T Y,et al. Epigenetic control of metastasis-associated protein 1 gene expression by hepatitis B virus X protein during hepatocarcinogenesis[J]. Oncogenesis, 2014,3:e88.
[11] SHONA J K,SHON B H,PARK I Y,et al. Hepatitis B virus-X protein recruits histone deacetylase 1 to repress insulin-like growth factor binding protein 3 transcription[J]. Virus Res, 2009,139(1):14-21.
[12] ARZUMANYAN A,FRIEDMAN T,KOTEI E,et al. Epigenetic repression of E-cadherin expression by hepatitis B virus X antigen in liver cancer[J]. Oncogene,2012,31(5):563-572.
[13] LIU L P,LIANG H F,CHEN X P,et al. The role of NF-kappaB in hepatitis B virus X protein-mediated upregulation of VEGF and MMPs[J]. Cancer Invest,2010,28(5):443-451.
[14] PAN J B,CLAYTON M,FEITELSON M A. Hepatitis B virus X antigen promotes transforming growth factor-beta 1(TGF-beta 1) activity by up-regulation of TGF-beta 1 and down-regulation of alpha(2)-macroglobulin[J]. J General Virol,2004,85:275-282.

An inducible expression system of LV-HBx for transducing primary human hepatocyte

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