[The comparative study on the regulation of apoptosis by hepatitis B virus X protein between B and C genotype].

Abstract

Objective: To investigate the apoptosis regulation on hepatoma cells by HBx between genotype B and C.

Methods: Genotype B and C HBx gene fragments were amplified and inserted into green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP-XB and pGFP-XC. The pEGFP-C1, pGFP-XB and pGFP-XC were introduced into Bel-7402 cells by Fugene HD to obtain Bel-7402 cells expressing GFP. The transcription and expression of HBx gene were demonstrated by RT-PCR and Western Blot analysis. Bel-7402, Bel-7402/GFP, Bel-7402/GFP-XB, Bel-7402/GFP-XC cells were treated with adriamycin (2.5 microg/ml), and the apoptosis of the cells was determined by trypan blue exclusion, and flow cytometry analysis.

Results: RT-PCR and Western Blot analysis showed that HBx genes of genotypes B and C were transcribed and expressed in Bel-7402/GFP-XB, Bel-7402/GFP-XC cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in Bel-7402, Bel-7402/GFP cells while no significant cell death was observed in Bel-7402/GFP-XB, Bel-7402/GFP-XC cells. Flow cytometry analysis indicated that no significant differences of apoptosis rates of Bel-7402/GFP-XB (3.87%) and of Bel7402/GFP-XC (4.01%) were observed (P > 0.05), moreover, no significant differences of Bel-7402/ GFP-XB (3.87%), Be17402/GFP-XC (4.01%) and of the untreated cells. Apoptosis rates in Bel-7402/GFP-XB (3.87%), Bel-7402/GFP-XC (4.01%) cells were significantly lower than those in Bel-7402 (27.05%) and Bel-7402/GFP (29.14%) cells at 48 hours after the adriamycin treatment (P < 0.01).

Conclusions: Bel-7402 cell lines expressing GFP, GFP-XB and GFP-XC fusion proteins were successfully established. HBV X protein blocks adriamycin-induced apoptosis of Bel-7402 cells. There is no difference between HBx of genotype B and C in inhibiting apoptosis induced by adriamycin.