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This dissertation, The Influences of Lead Ions on Viability, Proliferation and Neuronal Differentiation of Hippocampal-derived Neural Stem Cells of Newborn and Adult Rats by Yan-ho, Chan, 陳恩浩, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Neural stem cells (NSCs) are defined as multipotent stem cells. They are able to self-renew and differentiate into mature cells, such as neurons, oligodendrocytes and astrocytes. Neurotoxicity of lead (Pb2+) has been extensively investigated by many previous studies. These studies proved that lead is a potent toxin that affects nervous system, especially children's brain. However, most of these studies focused on the negative effects of lead on the differentiated or mature cell types in the brains instead of NSCs. The aim of this study was to reveal the effects of Pb2+ on viability, proliferation and differentiation of NSCs derived from the hippocampus of newborn rats aged 7 days and adult rats aged 90 days in vitro. NSCs harvested from the rat hippocampus were cultured in proliferation medium. After 6-8 days, free-floating neurospheres formed. The neurospheres were dissociated and plated onto poly-L-lysine coated 96-well plate and coverslips. Some dissociated cells were characterized by being stained with anti-nestin to show the presence of NSCs. This project was divided into three parts. In the first part, the Passage 2 (P2) cells plated onto 96-well plate were cultured in the proliferation medium with different concentrations of lead acetate (0-200μM) for 48 hours, followed by 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to detect the effects of Pb2+ on the cell viability. In the second part, P2-NSCs plated onto coverslips in wells were cultured in the proliferation medium with different concentrations of lead acetate (0-200μM). Then, 10 μM bromodeoxyuridine (BrdU) was added into the culture medium for additional 24 hours, followed by immunocytochemistry staining with anti-BrdU. In the last part, the dissociated P2-NSCs plated onto coverslips were allowed to grow in the differentiation medium of neurons, astrocytes or oligodendrocytes with different concentrations of lead acetate (0-200μM). After 6 days, immunocytochemistry staining with anti-microtubule-associated protein 2 (anti-MAP2), anti-glial fibrillary acidic protein (anti-GFAP) or anti-RIP was used to detect the differentiation commitment of affected NSCs. Low level of Pb2+ (1-10μM) had no effect on the viability of adult hippocampal neural stem cells (hNSCs). However, Pb2+ exposure at the concentration of 10μM could lead to significant cell death of newborn hNSCs. High level of Pb2+ (50-200μM) caused significant cell death of both newborn and adult hNSCs. Newborn hNSCs were sensitive to Pb2+ toxicity in proliferation assay. Even a low concentration (1μM) of lead could lead to significant inhibition of cell proliferation. High level of Pb2+ (50-200μM) suppressed proliferation of both newborn and adult hNSCs significantly. Moderate to high levels of Pb2+ exposure (50-200μM) significant decreased the percentage of mature neurons cultured from both newborn and adult hNSCs. Furthermore, 10μM or more Pb2+could significantly inhibited the oligodendrocyte differentiation of both newborn and adult hNSCs. However, Pb2+ could also stimulate the astrocyte differentiation of hNSCs. Lead concentrations higher than 10μM and 50μM could respectively lead to a significant increase in the percentage of mature astrocytes differentiated from newborn and adult hNSCs. The data showed that Pb2+ inhibited not only the viability

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