A Study On HBV Cultured In Human Cord Blood Mononuclear Cells

Objectives： To establish the vitro culture system of human cord bloodmononuclear cells,to understand the replication,secretion process of HBV in thehuman cord blood mononuclear cells by,and to establish a model of human cordblood mononuclear cells infected with HBV,which will lay the foundation forexploring and studying the molecular mechanism of HBV intrauterine infectionand HBV vertical infection.Methods： Cord bloods were collected from Obstetrics and Gynecologydepartment,affiliated hospital of Dali university,using EDTA as anticoagulant andhuman cord blood mononuclear cells were screened with Ficoll density gradientcentrifugation method,trypan blue staining showed that the living cell ratio was98%.The cord blood mononuclear cells were inoculated in24wells cell cultureplates with RPMI1640culture medium in accordance with the density of1×106/ml,cultured at37℃、5%carbon dioxide incubator and observed the dynamicchanges of cell morphology under the inverted microscope.Human cord bloodmononuclear cells were infected with the virus particles in a ratio of100:1，cultured for15days and everyday，the cell culture supernatant were tested for theHBV five indicators (HBsAg,HBsAb,HBeAg,HBeAb，HBcAb) by enzyme-linkedimmunosorbent assay(ELISA),fluorescence quantitative polymerase chainreaction(FQ-PCR) technology were conducted to quantitatively detect the HBVfrom cell culture supernatant and the intracellular and to determine the dynamicchanges of HBV DNA infection.Results：(1) Dynamic changes of the cord blood mononuclear cells morphology: Cord blood mononuclear cells cultured in15days maintained the normal cellmorphology and structure,most of the cell growed in suspension while a fewadherent to the wall.(2) ELISA assay to detect the Hepatitis B five indicators in the culture cellsupernatant:one day,HBsAg was positive in the first day,became negative in thesecond days then turned to be positive after cultured for7to10days and later becamenegative,while HBsAb,HBeAg,HBeAb and HBcAb were always negative.(3) Fluorescence quantitative PCR to detect HBV DNA: the cell culture mediumwas changed daily,and HBV DNA in cell culture supernatants could be detected inone day,not be detected after2to3days,while detected again in four days and thecopy number of HBV DNA increased gradually, in the9days,it presented thedowntrend and could not be detected in the13days, the curve of the copy number ofHBV DNA was close to the inverted "V"; and HBV DNA levels in the cell culturesupernatants without the replacement of the cell culture medium gradually increasedand the curve of HBV DNA copy number presented as "S";HBV DNA in culturedcells could be detected HBV DNA from the4days while not in the first1-3days,intracellular HBV DNA content were highest in the9days while it decreasedgradually with the increase of the number of cultured days and the curve of the copynumber of HBV DNAshowed as the inverted "V".Conclusion：(1) Having established a system of human cord blood mononuclear cellsin vitro；(2)Human cord blood mononuclear cells could be used as a cellular model ofHBV infection；(3) FQ-PCR method can be better than ELSIAto assay the HBV.