Hhv - 6 - A Latent Infection Model And The Influence On U251 Cell Cycle Progression

The incidence of primary central nervous system tumors is16.5/100,000. Gliomawhich accounts for about40%to50%of intracranial tumors is the most commonintracranial malignant tumor and ranks second in the children’s cancer. The incidencerate of glioma is3-6/100,000and the number of annual death is30,000in china.Glioma derives from neuroepithelial, which originated in the glial, neuralmesenchymal cells, ependymal choroid plexus epithelial and neurons. However, itsetiology and pathogenesis is unclear. In recent years, studies on epidemiological ofglioma found a variety of predisposing factors, such as genetic susceptibility,radioactive substances exposure, cancer genes, viral infections et al. HHV-6is adouble-stranded DNA virus which addict to human lymphocytes and glial cells, canestablish long-term latent infection in the body, can be repeated activation of viralgenes can be integrated into the host chromosome. The role of HHV-6in theoccurrence and development of glioma should be studied further.HHV-6serology positive rate was90%in healthy people. According to theantigenicity, host range, and nucleic acid restriction enzyme, HHV-6was divided intotwo subtypes, the homology of nucleic acid is approximate90%, variants A and B.HHV-6B has been identified as the causative agent of exanthem subitum, and alsorelated with lymphoproliferative disease. Knox, et al isolated HHV-6A from centralnervous system demyelinating areas of a patient with acquired immunodeficiencysyndrome in1995. The current study does not found that HHV-6A associated with aparticular disease.The tropism of HHV-6A and HHV-6B to nerve cell was different, and thetropism of HHV-6A to nerve tissue is stronger in vivo.The detection rate of HHV-6Ain the brain tissue is more than three times of HHV-6B, HHV-6A can generateinfectious virus particles in astrocytes, while HHV-6B. HHV-6A infectedoligodendrocytes can be cytopathic effect, persistence of DNA, and gradually into thelatent infection. There were lots of study about latent infection and tumor, such asHSV-1, HPV, HBV,et al. This study was to investigate HHV-6A infected U251cells into the latent infection after the acute infection, and the change and preliminarymechanism of proliferation and cell cycle in the process.Firstly, The GS strain of HHV-6infected HSB2which was identificated by PCRand immunofluorescence assay. HHV-6infected cells were collected and subjected tothe freeze-thaw cycle. The supernatant was concentrated by high speed centrifugation.The standard plasmid of pMD19T-U22was constructed, and then the virus titer wasdetermined by real-time PCR.Secondly, the cytopathic effect of HHV-6A GS infected U251cells wereobserved under light microscope. HHV-6U22gene was identified by PCR, HHV-6antigen expression was detected by immunofluorescence, and virus particles weredetected by electron microscopy, virus mRNA was detected by RT-PCR in theinfected U251cells. HHV-6gene copies were detected by real-time quantitative PCRin the infected U251cells and the supernatant. Results showed that HHV-6A couldinfect glioma U251cells which exited CPE, viral gene, viral particles and viralantigen expression. HHV-6DNA in the cell contentgradually reduced, and ultimatelyto maintain a low level with the increase in the algebra of passage.Lastly, the cell proliferation of HHV-6infected u251was detected by MTTassay; the cell cycle of HHV-6infected u251was detected by flow cytometry, themechanism of change of cell cycle was investigated by Western blot. Results showedthat HHV-6A promoted the proliferation, and the percentage of G1phase reducedwhile the percentage of S increased in the active infection. The total cyclinA andcyclinE1levels were increased, we found the protein levels of p21and Rb werereduced markedly; and the levels of cyclinD1and p53were basically unchanged inthe active HHV-6infection.In summary, the results demonstrate that HHV-6A can infect U251cells, and thelatent infection of HHV-6A can be established in the U251cells. HHV-6activeinfection increase s phase of cell cycle to promote the proliferation of glial cells. Theresults suggest that the relationship may exist between HHV-6infection and glioma, provide a relevant basis for further study of HHV-6in the occurrence anddevelopment of glioma.