Hepatitis B virus X protein suppressing adriamycin-induced apoptosis of HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the effect of hepatitis B virus X protein (HBx) on adriamycin-induced apoptosis of hepatocellular carcinoma cells.</p><p><b>METHODS</b>HBx gene fragment was amplified from subtype adr HBV plasmid by PCR, and inserted into Hind III and Kpn I sites of green fluorescent protein (GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP/HBx. The pEGFP-C1 and pGFP-HBx were introduced into HepG2 cells by Lipofectamine 2000 to obtain HepG2 cells expressing GFP. GFP-HBx fusion protein was selected using G418. The expression of HBx gene was demonstrated by RT-PCR analysis. HepG2, HepG2/GFP and HepG2/GFP-HBx cells were treated with adriamycin (2.5 microg/ml), and apoptosis of the cells was determined by their morphological changes, trypan blue exclusion, and flow cytometry analysis.</p><p><b>RESULTS</b>Under a fluorescence microscope, visible expression of GFP and GFP-HBx fusion proteins were observed in HepG2/GFP and HepG2/GFP-HBx cells, even after growing over 70 generations. RT-PCR analysis showed that HBx gene was expressed in HepG2/GFP-HBx cells. Trypan blue exclusion showed adriamycin induced time-dependent cell death in HepG2 and HepG2/GFP cells while no significant cell death was observed in HepG2/GFP-HBx cells. Flow cytometry analysis showed that apoptosis rates in HepG2/GFP-HBx (3.94%) cells were significantly lower than those in HepG2 (59.03%) and HepG2/GFP cells (61.38%) at 36 hours after the adriamycin treatment (P < 0.01). No significant differences of apoptosis rates of HepG2/GFP-HBx (3.94%) and of the untreated cells (2.12%, 2.78%, 2.55%) (P > 0.05) were observed.</p><p><b>CONCLUSION</b>A HepG2 cell line expressing GFP and GFP-HBx fusion proteins was successfully established. HBV X protein blocks adriamycin-induced apoptosis of these HepG2 cells.</p>

Detection of coxsackie virus B3 gene in myocardium and spleen samples in myocarditis with sudden death / 法医学杂志

OBJECTIVE@#To detect the Coxsackie virus B3(CVB3) gene in myocardium and spleen tissues in viral myocarditis(VMC) with sudden death and to explore the diagnostic method for VMC by means of seeking pathogene.@*METHODS@#By in situ RT-PCR, the detection of CVB3 gene in myocardium and spleen sections were performed in sudden death group caused by VMC and non-cardiac death group.@*RESULTS@#In VMC group, CVB3 gene-positive signals were seen in myocardium sections(3 out of total 8 cases, No. 1, 4, 7 cases) and spleen sections(4 out of total 8 cases, No. 2, 4, 6, 7 cases). In non-cardiac death group, no positive signals were detected in both myocardium and spleen tissues.@*CONCLUSION@#Positive detection of CVB3 gene in both myocardium and spleen maybe an important character of VMC and can improve the detecting pathogene in diagnosing VMC.