The Effect On Proliferation And Apoptosis Of Oligodendrocyte Cell Infected By Borna Disease Virus

Background and Objective:Borna disease virus (BDV), is a nonsegmented, negativesingle-stranded RNA virus. It is hightly neurotropic, which could causepersistent infection of the central nervous system(CNS). It has wide hostrange, and it could cause nature infection of warm-blood animals,showingthe obvious mental and behavioral disorders. Large number ofepidemiological studies have shown that BDV infection may be associatedwith the occurrence of human mental illness. BDV can infect neurons andglial cells, causing nerve cell plasticity, secretion of metabolic changes, butthe specific mechanism of infection reported less at home and abroad,especially BDV infection mechanism of glial cells is more rare.In this study, we used borna virus to infect human oligodendrocytes(OL), and compared the impact of BDV virus titers by the serumconcentration to expore the most appropriate serum concentration forinfection. In addition, we infected the OL cells in the appropriate serumconcentration to observe its impact on growth and apoptosis, and to explore the underlying molecular mechanisms.Methods:1We recovery OL/BDV Hu cell lines and obtained virus solutionby repeated freezing and thawing.Then we cultured OL cells in DMEMmediun containing different FBS concentrations and infected the cells withvirus solution of different dilution. After7days of culturing,we can useImmunohistochemical method to calculate the number of viruspositive cell colony,and find the most suitable serum concentrations forBDV infection.2We cultured the human oligodendrocyte Strain cell (OL cell) whichhad been established from human oligodendroglioma cell. OL cells wereinfected by BDV virus solution and identified by BDV nucleoprotein (P40protein) immunofluorescence.3Using CCK8kit, we measured the growth and proliferation of OLcells after BDV infection1to7days,and draw the growth curve. at differenttime points within24h after BDV infected OL cells,we use flow cytometry todetect cell cycle phases of DNA content and compar OL cell proliferationabilities betweenBDV infection group and the control group. We collect OLcells at1,3,5days after BDV infection and use flow cytometry to measurethe number of apoptotic cells, with the control group for statisticalcomparison.4we cultured cells, extracted OL cells protein at1,3,5days after BDV infection, detect apoptosis-related targets Bcl-2, Bax protein changes inrelative expression by Western-blot and analys the expression difference.Results:1After detecting virus titer,we can find that the virus titer value of2%FBS concentration was higher than the other concentrations.2By immunofluorescence we found that after3days and5days ofBDV infection th, P40protein expression of green fluorescence can befound in OL cell, indicating the success of BDV infection.3By CCK8kit determining cell proliferative capacity,we found thatwithin7days of infection, as time increased, the number of cells graduallyincreased. Compared with control cells, proliferative capacity of infectedcells is lower (P <0.05).4Measuring OL cell cycle within24hours after BDV infection byflow cytometry,we found G2phase of infected cells delayed and cellproliferation index reduced. The other hand,measuring the number ofapoptotic cells by flow cytometry in three days and five days of BDVinfection, the number of apoptotic cells significantly more than controlcells (P <0.05).5Detecting the relative expression levels of apoptosis related proteinsBcl-2and Bax by Western-blot method,we found after3days and5days ofinfection, the expression level of pro-apoptotic protein Bax wassignificantly higher than the control group; the level of antiapoptotic protein Bcl-2was significantly lower (P <0.05).Conclusion:1Different serum concentrations have impact on Borna disease virusInfectious Titer,2%FBS concentration may be better for the experiment ofBDV cell infection.2BDV infection could inhibit cell proliferation, and induce cellapoptosis increased through increasing Bax protein expression andresducing the Bcl-2protein expression.