人肝细胞的HBV感染和壳体化HBV DNA的量化

Kai Li; Seiichi Sato; Akinori Takaoka

doi:10.21769/BioProtoc.1717

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Peer-reviewed

HBV Infection in Human Hepatocytes and Quantification of Encapsidated HBV DNA

人肝细胞的HBV感染和壳体化HBV DNA的量化

KL Kai Li

SS Seiichi Sato

Akinori Takaoka email

发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1717 浏览次数: 10576

评审: Jia LiRamalingam BethunaickanAnonymous reviewer(s)

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Abstract

Human hepatic cancer cell lines such as HepG2, Huh7, and HLE cannot get infected with Hepatitis B virus (HBV) due to lack of an HBV receptor(s). Transfection with HBV genome has so far been referred as a tool to mimic HBV infection. However, since sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for HBV (Yan et al., 2012), hepatocyte cell lines that were stably transfected with a plasmid for NTCP expression have been used for HBV infection. This protocol is designed for infection with HBV in human hepatocyte cell line HepG2 expressing NTCP (HepG2-hNTCP-C4 cells; Iwamoto et al., 2014) or primary human hepatocytes (PHHs). In this section, we also describe one of the methods for the assessment of HBV infection: Quantification of the intracellular encapsidated HBV DNA.

Keywords: Hepatitis B virus (乙型肝炎病毒)

Hepatocyte (肝细胞)

Infection (感染)

Zhr (zhr)

Materials and Reagents

Materials

0.1-10 μl pipet tips (Thermo Fisher Scientific, catalog number: QSP#TF104 )
1-200 μl and 100-1,000 μl pipet tips (Corning, catalog numbers: 4845 and 4846 , respectively)
Falcon 12-well tissue culture plate (Corning, catalog number: 353043 )
Biocoat collagen I cellware 12-well plate (Corning, catalog number: 356500 )
1.5 ml and 2.0 ml microcentrifuge tubes (Corning, catalog numbers: MCT-150-A and MCT-200-C , respectively)
15 ml and 50 ml centrifuge tubes (Corning Incorporated, catalog numbers: 352096 and 352070 , respectively)
96-well fast plate (NIPPON Genetics, catalog number: 38801 )

Reagents

Hepatocyte culture and infection with HBV
1. Primary human hepatocytes (PHHs) (PhoenixBio Co.)
2. HepG2-hNTCP-C4 cells (Drs. Takaji Wakita and Koichi Watashi, Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Iwamoto et al., 2014)
3. Dulbecco’s Modified Eagle’s Medium (DMEM) (NISSUI PHARMACEUTICAL, catalog number: 05919 )
4. DMEM/F-12+GlutaMax (Life Technologies, catalog number: 31331-028 )
  Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 31331-028 ”.
5. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Life Technologies, catalog number: 15630-080 )
  Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 15630-080”.
6. Fetal bovine serum (FBS) (Life Technologies, catalog number: 10437-028 )
  Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 10437-028”.
7. G418 (Nacalai tesque, catalog number: 09380-86 )
8. Phosphate-buffered saline (PBS) (pH 7.4)
9. HBV plasmid (pUC19-HBV, genotype C) (Dr. Yasuhito Tanaka, Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; Sugiyama et al., 2006)
10. FuGENE 6 transfection reagent (Promega Corporation, catalog number: E2692 )
11. Opti-MEM I reduced-serum medium (Life Technologies, catalog number: 31985-070 )
  Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 31985-070”.
12. QIAamp DNA Blood Mini Kit (QIAGEN, catalog number: 51104 )
13. Polyethyleneglycol 8000 (PEG 8000) (Sigma-Aldrich, catalog number: 81268 )
14. PHHs culture media (see Recipes)
15. HepG2-hNTCP-C4 culture media (see Recipes)
Encapsidated HBV DNA extraction
1. Nuclease free-H₂O
2. Tris (hydroxymethyl) aminomethane (Tris) (Nacalai tesque, catalog number: 35406-91 )
3. NP-40 (Nacalai tesque, catalog number: 25223-75 )
4. Magnesium acetate (MgOAc) (Wako Pure Chemical Industries, Siyaku, catalog number: 130-00095 )
5. Ethylenediaminetetraacetic acid (EDTA) (Wako Pure Chemical Industries, Siyaku, catalog number: 345-01865 )
6. Proteases K (Life Technologies, catalog number: 25530-015 )
  Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 25530-015”.
7. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, Siyaku, catalog number: 191-07145 )
8. Sodium chloride (NaCl) (Nacalai tesque, catalog number: 31320-05 )
9. Deoxyribonuclease I (DNase I) (Promega Corporation, catalog number: M6101A )
10. Ribonuclease A (RNase A) (Life Technologies, catalog number: 12091-021 )
  Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: 12091-021”.
11. Phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich, catalog number: P2069 )
12. Chloroform (KANTO CHEMICAL, catalog number: 07278-00 )
13. Sodium acetate (NaOAc) (Wako Pure Chemical Industries, Siyaku, catalog number: 198-01055 )
14. Glycogen (Roche Diagnostics, catalog number: 10901393001 )
15. Isopropanol (Nacalai tesque, catalog number: 29113-53 )
16. Ethanol (99.5%) (Nacalai tesque, catalog number: 14713-95 )
17. Lysis buffer (see Recipes)
qPCR for quantification of HBV DNA
1. SYBR Premix Ex Taq (2x) (Tli RNase H Plus) (Takara Bio Company, catalog number: RR420 )
2. ROX reference dye (50x) (Takara Bio Company, catalog number: A9701A )
3. Primers for amplification of encapsidated HBV DNA by quantitative PCR
  Forward: 5’-CTTCATCCTGCTGCTATGCCT-3’
  Reverse: 5’-AAAGCCCAGGATGATGGGAT-3’
  (Product length: 222 bp, product Tm: 83.5 °C)

Equipment

37 °C and 5% CO₂ cell culture incubator (WAKENBTECH CO., catalog number: 9000EX )
Pipettes (PIPETMAN P2, P20 and P1000) (Gilson Scientific, catalog numbers: F144801 , F123600 and F123602 , respectively)
High speed refrigerated micro centrifuge (KUBOTA Corporation, catalog number: 3500 )
Vortex mixer (Labnet Internationa, catalog number: vx100 )
Shaker (Tokyo Rikakikai Co., EYELA, catalog number: MMS-110 )
Double aluminum block bath (SCINICS CORPORATION, catalog number: ALB-301 )
ABI StepOnePlus™ Real-Time PCR Systems (Life Technologies, catalog number: 4379216 )
Note: Currently, it is “Thermo Fisher Scientific, Applied Biosystems™, catalog number: 4379216”.
qPCR adhesive seal (NIPPON Genetics, catalog number: 4Ti-0560 )

Procedure

English

中文翻译

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如何引用

Li, K., Sato, S. and Takaoka, A. (2016). HBV Infection in Human Hepatocytes and Quantification of Encapsidated HBV DNA. Bio-protocol 6(2): e1717. DOI: 10.21769/BioProtoc.1717.