1α-hydroxylase, 25-hydroxyvitamin-D 1α-hydroxylase; 1α,25(OH)2D,

1α-hydroxylase, 25-hydroxyvitamin-D 1α-hydroxylase; 1α,25(OH)2D, 1α,25-dihydroxyvitamin D; 24-hydroxylase, Protein Tyrosine Kinase inhibitor 25-hydroxyvitamin-D 24-hydroxylase; 25(OH)D, 25-hydroxyvitamin D; HCV, hepatitis C virus; IFNα, interferon-α; ISG, interferon-stimulated gene; SVR, sustained viral response; TLR3, Toll-like receptor 3; RIG-I, retinoic acid-inducible gene I; VDR, vitamin D receptor. Vitamin D3 was purchased from Sigma Chemical (St. Louis,

MO). Calcitriol was obtained from Teva Pharmaceutical Industries (Israel). Virus assays were carried out with the intergenotypic HJ3-5 chimeric HCV virus.25 Virus stocks were produced in FT3-7 cells.26 Huh-7.5 cells were used for all assays and were cultured as described.25 Total RNA was extracted from cells using the guanidine isothiocyanate method.27 RNA samples were treated with DNaseI (Ambion, Cambridgeshire, UK). Total RNA (1 μg) was subjected to reverse transcription (RT) using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real-time RT-PCR assays were performed in the ABI 7000 sequence device (Applied Biosystems), using TaqMan selleck chemicals llc gene expression assays essentially as described.28 For measurement of HCV expression, 20-μL reactions were prepared in a 96-well

format, using 1 μL of the template cDNA, 10 μL of 2× TaqMan Universal PCR Master (Applied Biosystems), 10 pmol of forward primer HCV-F (nucleotides [nt] 130-146), and reverse primer HCV-R (nt 272-290) (AF139594) for the HCV nontranslated region, and 5 pmol of an HCV-specific TaqMan probe. Assay-on-Demand Gene Expression Products (Applied Biosystems) were 上海皓元医药股份有限公司 used for the measurement of IFN-β (Hs02621180_s1), GAPDH (Hs99999905_m1) CYP27B1 (Hs00168017_m1), CYP24A1 (Hs001679999_m1), and VDR (Hs00172113_m1). Results are expressed as the ratio of the target gene messenger RNA (mRNA) and GAPDH

mRNA threshold values. Analysis of the ISG MxA expression level was performed by SYBR Green I dye (Applied Biosystems) using MxA-specific primers as published29 and GAPDH as the internal control gene (primers: GAPDHS 5′-GAAGGTGAAGGTCGGA GTC-3′ and GAPDHAS 5′-GAAGATGGTGATGG GATTTC-3′) using the ABI 7000 (Applied Biosystems) detection system. The level of calcitriol in the supernatants was determined by specific 1α,25-dihydroxyvitamin D enzyme-linked immunosorbent assay (ELISA) kit following extraction with monoclonal anti-1α,25-dihydroxyvitamin D antibody according to the manufacturer’s instructions (Immunodiagnostic Systems, Boldon, UK). Huh-7.5 cells were pretreated with vitamin D3, calcitriol, or the vehicle ethanol (ethanol concentration did not exceed 0.015%) for 3 hours before infection with the HJ3-5 virus at a multiplicity of infection (moi) of 0.1-0.01.

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