Preliminary Study Of Proliferation And Apoptosis Of Human Astrocytoma Cell Affected By Borna Disease Virus Hu-H1 Strain

BACKGROUND:Borna disease virus (BDV), is a nonsegmented, negative-sense, single-strand (NNS) RNA virus. BDV is highly neurotropic and has a non-cytolytic strategy for replication in cells and replicates and transcribes in the nucleus of infected cells. BDV can experimentally infect many vertebrates from rabbits and monkeys to chickens, and causes neurological symptoms. Epidemiological studies have demonstrated that natural infection of BDV has been found in a wide variety of hosts. Up to present, numerous studies have documented that BDV can infect humans. Furthermore, isolation of human BDV from the peripheral blood or brain of neuropsychiatric patients has been reported from several countries. The precise mechanism how BDV induced the loss of neurons and impaired the brain function development is still unknown.Objective:In this study, we try to infect U87 cell with BDV-Hu-H1 and determine the proliferation and apoptosis after infection. So we can have a basic knowledge of the interaction between U87 cell and Hu-Hl.Methods:1. To obtain the virus stock by repeated freeze-thawing cycle.2. To quantify the virus titer via immunocytochemical assay.3. We detected the proliferation of infected and uninfected U87 cell with CCK8 kit from 1 to 7 day postinfection.4. To detect the apoptosis proportion of infected and uninfected U87 cell with Annexin V-FITC apoptosis detection kit at 1,3,5d postinfection, respectively.5. To detect cell cycle delay of BDV-infected U87 cell by flow cytometry at time points of Oh,6h,15h, and 24h.6. We detected apoptosis-related protein Bcl-2 and Bax level of infected and uninfected U87 by western blot.Results:1. The titer of Hu-H1 was 6×10^4FFU/mL.2. The expression of p40 protein could be detected at 3 day postinfection. Hu-H1 could infect U87 cell efficiently.3. There was no difference between BDV group and Control group at 1d p.i. The proliferation of BDV group was lower than Control, and BDV-infected U87 grew slower at 2-4d p.i. BDV-infected U87 cell grew faster than Control at 5 and 6d p.i.4. There was no difference of apoptosis proportion between BDV group and Control group at Id p.i. The apoptosis of BDV group was higher than Control at 3d p.i. and was lower than Control at 5d p.i.5. Serum starvation could delay the cell cycle and there was no difference between BDV and Control group. At 6h,15h,24h p.i., the proliferation index of BDV group was lower than Control, which indicated Hu-H1 hampered cell growth at this time.6. At 1d p.i., the level of neither Bcl-2 nor Bax had no difference. At 3d and 5d, the level of Bcl-2 in BDV group was lower than.Control, whereas the level of Bax was higher than Control.Conclusion:In this study, Hu-Hl infected U87 cell efficiently and affected the proliferation and apoptosis. In the early phase, BDV inhibited the proliferation and promoted the apoptosis. However, in the later phase, the impact was revered totally.