Borna Disease Virus Nucleoprotein Regulation Of Neural Stem Cell Survival, Proliferation And Differentiation Of The Preliminary Study

BACKGROUND AND OBJECTIVEThe neural stem cells (NSCs) grow and proliferate in the way of neurospheres cultured in vitro, while many experiments in studying of NSCs work on single cells. Therefore, it is imperative to dissociate neurospheres to single cells safely and efficiently. Now there are various methods of dissociation with omnigenous defects in safety and effectiveness. The purpose of this study is to explore a safe and effective method for dissociating neurospheres cultured in vitro.METHOD1. NSCs were isolated from hippocamps of new-born Sprague-Dawley (SD) rats (younger than 24h), cultured in vitro, identified by nestin immunofluorescent staining.2. NSCs are divided into 4 groups and the neurospheres of each group were dissociated using the 4 methods respectively, (1) digestion of trypsin,(2)triturating only with pasteur pipette,(3)grinding with stainless steel mesh, (4) controlling nerospheres size combining short-time trypsin digestion.3. Neurospheres'dissociation effeciency and growing situation were observed under the phase contrast microscope in 5min and 1d after dissociation.4. Trypan blue staining was used to count the alive cells and calculate the survival rates in 5min and 1d after dissociation.RESULTThough prolonging trypsin digestion time led to dissociation of neurospheres(>30min),NSCs were hardly survival. Neurospheres could not be dissociated completely by triturating only with pasteur pipette. Grinding with stainless steel mesh would damage neurospheres severely which result in low NSCs survival rates. In contrast, controlling nerospheres size combining short-time trypsin digestion could dissociate neurospheres better than the other 3 methods,and have a significantly higher NSCs survival rate(5 min 92.2% and 1d 82.0% after dissociation, p<0.05).CONCLUSIONcontrolling nerospheres size(diameter is about 50μm) combining short-time (about 5min) trypsin digestion could be a safe and effective method of dissociating neurospheres. BACKGROUND AND OBJECTIVEBorna Disease Virus(BDV)is a neurotropic virus and its infection was found being involved in human neurological and psychiatric disorders including depression in various researches, while its pathogenesis is unclear. BDV nucleoprotein is one of the main proteins of BDV coded by p40 genetic fragment as the most abundant protein in the cell with the smallest variation. Some studies indicated that extracellular signal regulated kinase1/2(ERK1/2) signal cascade plays a crucial role in survival, proliferation and differentiation of neural stem cells (NSCs). The purpose of this study is to transfect the eukaryotic expression plasmid containing BDV p40 gene,pEGFP-N1-p40, into NSCs derieved from hippocamps of new-born Sprague-Dawley (SD) rats, observe the variation of survival, proliferation, differentiation and ERK1/2 signal cascade of NSCs, and investigate the effect of p40 gene to NSCs and part of pathogenesis of BDV inducing neurological and psychiatric disorders. METHOD1. pEGFP-N1-p40 and pEGFP-N1 plasmids were transfected into NSCs using the cationic liposome respectively. Transfection efficiency was observed under the fluorescence microscope and BDV p40 expression in NSCs was indetificated by RT-PCR.2. Three groups were set up: non-transfection group, pEGFP-N1 transfection group and pEGFP-N1-p40 transfection group. Survival of NSCs was detected using CCK-8 kit, while proliferation was detected in BrdU uptaking experiment. The ratios of differentiation to neuro, astrocyte and oligodendrocyte were investigated in the 14th day after adherent differention by immunohistochemical staining. Westernblot was used to detect the activation of ERK1/2 of NSCs .RESULT1. NSCs expressing BDV p40 were formed. Green fluorescence can be seen in the Cytoplasm and nuclei of about 10% NSCs of pEGFP-N1 or pEGFP-N1-p40 transfection group. It was indicated that BDV p40 genetic fragment was transcribed only in NSCs of pEGFP-N1-p40 transfection group by PCR result.2.(1) BDV p40 controlled the survival of NSCs. (2) BDV p40 restrained the proliferation of NSCs. (3) The sigificant difference of ratios of differentiation to neuro,astrocyte and oligodendrocyte in the 14th day after adherent differentiation wasn't indicated. (4) BDV p40 down-regulated ERK1/2 protein expression of rats NSCs. METHOD CONCLUSIONBDV p40 could restrain the survival and proliferation of NSCs, but have no significant effect on the direction of differentiation. BDV p40 could down-regulate ERK1/2 protein expression of NSCs. It would be possible that BDV p40 restrained the survival and proliferation of NSCs by down-regulating ERK1/2 activation of rats NSCs.