| Background Alzheimer's disease(AD)is the most common degenerative disease of the central nervous system,with the decline of cognitive function and activity of daily living as the main clinical manifestation.Psychological and behavioral symptoms can occur at different degree,and disabilities and loss of self-care are common in the later stage of AD.So,a heavy burden has been put on patients,families and society.However,there is still a lack of effective treatment to AD.Human umbilical cord mesenchymal stem cells(hUC-MSCs)have the potential for self-renewal,significant proliferation and high differentiation.hUC-MSCs can differentiate into neurons,glial cells,and also have an easily-separated and abundant source,low immunogenicity,immunomodulatory,paracrine,non-tumorigenic and limited ethical issues,so they are widely used in stem cell replacement therapy.Brain-derived neurotrophic factor(BDNF)is the most common neurotrophic factors in the brain and plays an important role in the survival and activity of neurons.Exogenous BDNF enhances the neuroprotective potential of MSCs in in vitro and in vivo,but the effect of BDNF transfected into hUC-MSCs-derived cholinergic-like neurons remains unclear.Objective To investigate the cholinergic activity in hUC-MSCs-derived cholinergic-like neurons transfected with GFP-BDNF lentivirus in vitro.Methods The mesenchymal stem cells in neonatal umbilical cord tissue of cesarean section were isolated and cultured by tissue block method.hUC-MSCs at the third passage(P3)were induced to differentiate into cholinergic-like neurons.Immunofluorescent staining was used to identify the expression of Ch AT,Nestin and the co-expression of Ch AT and MAP-2 before and after induction.The hUC-MSCs-derived cholinergic-like neurons were transfected with green fluorescence protein(GFP)-null vector lentivirus and GFP-BDNF lentivirus,respectively.The experiments were divided into four groups: hUC-MSCs group,cholinergic-like neurons group,and cholinergic-like neurons+null vector group,cholinergic-like neurons+overexpressed BDNF vector group.Immunofluorescent staining was used to identify GFP expression and observe the co-expression of Ch AT and MAP-2.Western blot(WB)was used to detect the expression of Ch AT,Nestin and BDNF in each group.Acetylcholine(Ach)content in cell culture supernatants was detected by acetylcholine kit.Results 1.hUC-MSCs can be induced to differentiate into cholinergic-like neurons.Immunofluorescent staining showed that the expression of Ch AT and Nestin after induction increased significantly than that before induction(P<0.01),and the expression of double-labeled Ch AT and MAP-2 increased significantly after induction.2.Immunofluorescent staining identification of GFP expression in each group.The expression of GFP was observed in cholinergic-like neurons + null vector group and cholinergic-like neurons+overexpressing BDNF group.3.Immunofluorescent staining identification of co-expression of Ch AT and MAP-2 in each group.The results showed that the co-expression of Ch AT and MAP-2 in cholinergic-like neurons+overexpressed BDNF group increased significantly compared with the other three groups.The co-expression of Ch AT and MAP-2 in cholinergic-like neurons+null vector group and cholinergic-like neuron group was significantly higher than that in hUC-MSCs group.4.Western blot results showed that the expression of Ch AT,Nestin and BDNF in cholinergic-like neurons+overexpressing BDNF group was significantly higher than those in other three groups(P<0.01).The expression of Ch AT,Nestin and BDNF in cholinergic-like neurons+null vector group and cholinergic-like neuron group was significantly higher than that in hUC-MSCs group(P<0.05).5.Ach content determination showed that Ach content in cell culture supernatant in cholinergic-like neurons+overexpressing BDNF group was significantly higher than those in other three groups(P<0.01),and Ach content in cholinergic-like neuron+null vector group and cholinergic-like neuron group was significantly higher than that in the hUC-MSCs group(P<0.05).Conclusions 1.hUC-MSCs can be induced to differentiate into cholinergic-like neurons.2.The expression of Ch AT,Nestin and the co-expression of Ch AT and MAP-2 increased in hUC-MSCs-derived cholinergic-like neurons transfected with GFP-BDNF lentivirus.3.hUC-MSCs-derived cholinergic-like neurons transfected with GFP-BDNF lentivirus promoted neurons to secrete acetylcholine.Background Alzheimer's disease(AD)is an age-related neurodegenerative disease and the most common type of dementia.The main pathological changes are senile plaques,neurofibrillary tangles and neuronal loss.There is no cure for AD yet.hUC-MSCs are pluripotent stem cells derived from human umbilical cord Walden's gel tissue and have multiple differentiation potentials.They can differentiate into cholinergic neurons in a specific environment and promote the release of acetylcholine.BDNF also promotes neurogenesis and synaptic formation,reducing oxidative stress and cell death.However,the role of BDNF modified hUC-MSCs-derived cholinergic-like neurons in AD rats remains unclear.Objective To investigate the therapeutic effects of BDNF modified hUC-MSCs-derived cholinergic-like neuronal transplantation in AD rat models.Methods Rats were randomly divided into AD model group and sham operation group.AD rats were injected with 1?L A?1-42 into the hippocampus region of the rat model group,and the same amount of saline was injected into the hippocampus region of the sham operation group.The hUC-MSCs-derived cholinergic-like neurons-null vectors cells and hUC-MSCs-derived cholinergic-like neurons-overexpressing BDNF vectors cells were slowly injected into the hippocampus of AD rats.The experiments were divided into four groups: the sham operation group,AD group,AD+cholinergic-like neurons-null vector group,AD+cholinergic-like neuron-overexpressed BDNF vector group.After 8 weeks of transplantation,Morris water maze test was performed to detect changes in spatial learning and memory ability of rats.Western blot was used to detect the expression of Ch AT,ACh E,BDNF and DCX in hippocampus.Immunohistochemistry was used to detect the expression of A? and BACE1 in hippocampal CA1,CA2,CA3 and DG regions.Immunofluorescence assay was done for the expression of astrocyte markers GFAP and microglia marker Iba-1.TUNEL detection was applied for neuronal apoptosis.Results 1.Morris water maze test showed that rats in the AD+cholinergic-like neurons-overexpressing BDNF vector group in the positioning navigation experiment significantly reduced the escape latency than those in the AD+cholinergic-like neurons-null vector group and the AD group(P<0.01).In the space exploration experiment,the percentage of time in the target quadrant and cross platform number in rats of the AD+ cholinergic-like neurons-overexpressing BDNF vector group were significantly higher than those in the AD+cholinergic-like neurons-null vector group and the AD group(P<0.01).2.Western blotting results showed that the expression of Ch AT,ACh E,BDNF,and DCX in the hippocampus of the AD+cholinergic-like neurons-overexpressing BDNF vector group rats was significantly increased than that of the AD+cholinergic-like neurons-null vector group and the AD group rats(P<0.01).3.Immunohistochemistry assay showed that the expression of A? and BACE1 in the CA1,CA2,CA3,DG area of the AD+cholinergic-like neurons-overexpressing BDNF vector group rats was significantly reduced than that of the AD+ cholinergic-like neuron-null vector group and the AD group rats(P<0.05).4.Immunofluorescence staining showed that the expression of astrocyte markers GFAP and microglia marker Iba-1 in the hippocampal brain tissue of the AD+cholinergic-like neurons-overexpressing BDNF vector group rats increased significantly than that of the AD+cholinergic-like neurons-null vector Group and the AD group rats(P<0.01).5.TUNEL detection showed that neuronal apoptosis in the hippocampus region of the AD+cholinergic-like neurons-overexpressed BDNF vector group rats was significantly reduced than that of the AD+cholinergic-like neurons–null vector group and the AD group rats(P<0.01)..Conclusions 1.Transplantation of BDNF modified hUC-MSCs-derived cholinergic-like neuronons could significantly improve spatial learning and memory abilities of AD rats.2.Compared with the hUC-MSCs-derived cholinergic-like neurons-null vector group and the AD group,rats in the AD+cholinergic-like neurons-overexpressed BDNF vector group increased the release of acetylcholine and Ch AT expression in the hippocampus.3.Compared with the hUC-MSCs-derived cholinergic-like neurons-null vector group and the AD group,rats in the AD+cholinergic-like neurons-overexpressed BDNF vector group significantly improved the activation of astrocytes and microglia and reduced the expression of A??BACE1.4.Compared with the hUC-MSCs-derived cholinergic-like neurons-null vector group and the AD group,rats in the AD+cholinergic-like neurons-overexpressed BDNF vector group significantly inhibited neuronal apoptosis,promoted neurogenesis and increased the expression of synapse-associated proteins. |
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