| Background: Alzheimer’s disease (AD) is a common neurodegenerative disordersin elderly people, which is characterized by progressive and irreversible decline ofmemory and cognitive function. The major pathological features include the progressivedegeneration and loss of cholinergic neurons and synapses encompassing the brain.Neural stem cell (NSC) is important pluripotent stem cells, which can differentiate toform the specified neurons, glial cells, and oligodendrocytes. NSC has potentialapplication for cell replacement therapy, such as AD, which is characterized byuncontrolled cell death. NSC can be cultured in vitro as neurospheres or adherentmonolayer, or be transferred neurospheres to monolayer culture. The mechanism duringdifferentiation to neurons is very complex and influenced by many factors. The presentstudy showed that nerve growth factor (NGF) and brain-derived neurotrophic factor(BDNF) BDNF treatment could support the survival of existing neurons as well asencourage the growth and differentiate of new neurons. NGF and BDNF involved inregulationg of NSC proliferation and diffiretiation via binding Trk receptors andactivating MAPK/ERK signal pathway. A basic helix-loop-helix (bHLH) is a proteinstructural motif that characterizes a family of transcription factors (TFs), which playsprominent roles in proliferation, growth and differentiation processes of NSC. HES1andHES5maintain the number and status of undifferentiated NSC and neural progenitors(NPs) cells and inhibite differentiation. MASH1, NGN1and NeuroD can promote neuraldifferentiation. The major goal to understand the differentiation mechanism of NSC isthat NSC is applied as transplation therapy to treat neurodegenerative diseases. It wasshowed that NSC could impreove function of impaired nerve after grafted and that NGFor BDNF treated NSC was improved in survival number and migration area. PartⅠ Culture of neural stem cells from rat embryo in vitroAims: Culturing of NSC in vitro was investigated, in which NSC could proliferateand differentiate. Methods: The cerebrums of rat embryos were separated in sterileworking condition and cultured in serum-free medium. When the secondary neurosphereswere formed, Accutase digested them into single cells. Then they were divided into twoparts, one was cultured with suspension neurospheres, the other was plated to adherentmonolayer. And the abilities of “stem” and “differentiation” in two methods wereexamined. Results: NSC I s obtained in both of two. Proportation of Nestin-positive cellsin adherent monolayer culture is93.17±3.06%. β-tubulin Ⅲ-positive cells is higher inadherent monolayer culture (19.55±1.09%) than in suspension neurospheres (11.91±2.73%) without any inducing factor. Conclusion: The adherent monolayer culture transferredfrom P2neurospheres is a better method for differentiation of neurons from NSC. PartⅡ Combined effect of NGF and BDNF on the neuronal differentiation ofneural stem cells and the potential molecular mechanismsAims The effect of NGF, BDNF and BNDF combined with NGF on neuronaldifferentiation of NSC and their possible mechanisms were investigated by this study.Methods Adherent monolayer culture was employed to obtain highly homogeneous NSC.The cells were divided into four groups: control, NGF, BDNF, and a combination(BDNF+NGF) group. The proportions of neuron and phospho-ERK (p-ERK) levels wereexamined using immunocytochemistry and western blotting. Q-PCR was used to measurethe mRNA levels of HES1, HES5, MASH1, NGN1and NeuroD at different timeintervals after neurotrophins-induced differentiation. Results Both NGF and BDNF arefound to induce the neuronal differentiation of NSC. Proportion of β-tubulin III-positivecells are31%in NGF,35%in BDNF and39%in combination at3days afterNTs-induced differentiation, the differences among them are statistically significant(P＜0.01). P-ERK expression levels are the higher in NGF and BDNF and highest incombination group, and differences among them are statistically significant(P＜0.05). Q-PCR results show that relative quantity (RQ) of HES1and HES5are evidently reducedafter NTs-induced differentiation. There is no significant difference among control, NGF,BDNF and combination group at all time points. Expressions of MASH1, NGN1andNeuroD are markedly enhanced in NTs-induced differentiation groups as compared to thecontrol and highest in combination group at all time intervals. The differences amongthem are statistically significant(P＜0.05). Conclusion β-tubulin Ⅲpositive neurons indifferent groups are positively correlated to the expression of MASH1, NGN1andNeuroD, and the level of p-ERK. BDNF combined with NGF significantly improvesneuronal differentiation of NSC, which may provide promising support for practicalapplication of NSC in neurodegenerative diseases. Part Ⅲ Combined effect of NGF and BDNF and NSC on rat model of Alzheimer’sdisease with192-IgG-saporinAims Effects of different mixed cells transplantation on model of Alzheimer’s diseasewith192-IgG-saporin were investiged. Methods Rats were divided into five groups:sham, AD model, NGF, BDNF and combination.4w after injection of AD model, ratsreceived mixed cells into the basal forebrain. Morris water maze andimmunohistochemistry were employed to test learning and memory, number ofcholinergic neuron and OD of synaptophysin. Histochemistry was employed to test lenthof nerve fibre. Results Learning and memory ability of AD model are significantlydeclined. Transplantation of mixed cells could significantly increase the ability, includingescape latency shorten (8.87±0.26s), frequency through the platform in spatial probeincreased (5.33±1.15), number of cholinergic neuron supplied (98.16%), OD ofsynaptophysin rescued (92.45%) and quantity of nerve fibre growth (95.41%). Thedifferences between cells transplantation group and AD model were statisticallysignificant(P＜0.05). Conclusion Nerve cells after induced3d by NGF combined BNDFware grafted into AD models. Learning and memory ability were significantly improved, which could be attributed to the rescue of cholinergic neuron, synaptophysin and nervefibre. Combination group had a more significantly effect than NGF or BDNF alone. Summary:1. The adherent monolayer culture transferred from P2neurospheres was a bettermethod for differentiation of neurons from NSC.2. β-tubulin Ⅲpositive neurons in different groups were positively correlated to theexpression of MASH1, NGN1and NeuroD, and the level of p-ERK. BDNF combinedwith NGF significantly improved neuronal differentiation of NSC, which may providepromising support for practical application of NSC in neurodegenerative diseases.3. Nerve cells after induced3d by NGF combined BNDF ware grafted into ADmodel. Learning and memory ability were significantly improved, which could beattributed to the recovery of cholinergic neuron, synaptophysin and nerve fibre.Combination group had a more significantly effect than NGF or BDNF alone. |
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