Enhancement of Survival， Proliferation and Differentiation into Neurons of Neural Progenitor Cells Isolated from Rats Hippocampus by Brain Derived Neurotrophic Factor

Abstract: The objective of the study is to explore the effect of brain derived neurotrophic factor (BDNF) on the neural progenitor/stem cells (NPCs) of rat hippocampus. NPCs were obtained from Wistar rats at embryonic day 15-16 and cultured in serum-free medium， then the neurospheres were harvested， fixed and immunostained for nestin (the protein marker of stem cells); NPCs were induced to differentiate with 1% fetal calf serum containing medium; The single NPC was obtained by a mechanical dissociation and grown for 3 days in medium containing BDNF at the concentration of 10?00 ng/ml then the number of neurosphere were counted and the diameter of neurosphere were measured; Tunel staining positive cells and the level of lactic acid dehydrogenase (LDH) in culture medium were used to evaluate the effect of BDNF on the NPCs survival; β tubulin III (Tuj-1) immunostaining was used to label the neurons differentiated from NPCs， and the percentage of Tuj-1 positive cells among DAPI positive cells in the BDNF and control group were compared. Additionally， the length of neurite in Tuj-1 immunostaining positive cells was measured. The results showed that nestin-positive NPCs exhibited the ability of self-proliferation and could be induced to differentiate into neurons and astrocytes. BDNF at the concentration of 10-200 ng/ml enhanced the proliferation of NPCs at the cell density of 5×10⁵ cells/ml， while 40 ng/ml BDNF exhibited the strongest proliferation enhancement; Diameter of newly formed neurosphere with 40 ng/ml BDNF was increased obviously while the apoptosis rate (Tunel⁺/DAPI⁺) and LDH leakage of 40 ng/ml BDNF group was significantly decreased compared with the control group. In 40 ng/ml BDNF group， more neurons (Tuj-1⁺ cells) were observed and total neuritic length in Tuj-1⁺ cells were significantly longer， compared to the control group. Our study suggest that BDNF has a neuroprotective effect， can promote NPCs proliferation and induce them differentiation into neurons.
    

CSTR: 32200.14.cjcb.2008.03.0022