Immune Regulatory Role Of Amniotic Mesenchymal Cells Against Neuronal Apoptosis Induced By β-amyloid Activated Microglia

Background Alzheimer’s disease (AD) is a commonly degenerative disease of the centralnervous system of characterized by progressive impairment of cognitive function andmemory of damage in the elderly. The etiology of AD is complicated as well as unknownpathogenesis. Now, most studies show that beta-amyloid protein plays an important role inAlzheimer’s disease. As chronic excitor substance, deposition of Aβgradually accumulateas neuritic plaques in the brain of AD. It can mediate microglia focal non-specificinflammatory reactions, and thus lead to neurons damage or death. So, The importantreason of neuronal apoptosis in AD maybe the deposition of β-amyloid activated microglia.Since AD pathogenesis is unclear, the treatment of AD is extremely difficult. Thetraditional treatment of AD was drug treatment, but the effect of drug treatment can onlyalleviate the symptoms of AD, does not stop the progress of this disease. Nowadays, withthe development and application of high technology，especially stem cell transplantationtechnology continues to improve, stem cell therapy brings new strategies and opportunitiesfor the treatment ofAD.The study found that Amniotic mesenchymal cells from the fetal membranes not onlyhas the characteristics of stem cells, weak immunogenicity, but itself hasimmunomodulatory properties, which can inhibit the proliferation of immune cells withoutcausing immune responses. AMSCs can inhibit excessive activation of the central nervoussystem of immune cells-MI and regulate a series of inflammatory cascade reaction. Giventhe pivotal role of microglia in AD, it is possible for AMSCs to had a protective effect onneuronal cell.Objective In this study, we aimed to analyze the stem cell properties of Amniotic mesenchymal cells and its immunosuppressive action on Aβ activated microglia. Next, weestablished a co-culture of microglia and AMSCs in vitro, and further explored that theactivation of MI mediated by Aβ can induce neuronal apoptosis, but, AMSCs may play aprotective role to neuronal by negatively regulating the activation of MI.Method1. The primary culture and identification of three kinds of cellsTo divide and culture AMSCs, MI and neurons in useing primary cell culturetechnology; Then to analyze the phenotype and biological properties withimmunofluorescence, flow cytometry,2. To build a co-culture system ofAMSCs and MIAMSCs and MI are divided into3groups with the same concentration of1.0×106/ml.(1) Control group: MI are cultured only.(2)Activated byAβ1-42group: Aβ1-42is added in cultured MI(3) Co-cultured group:Aβ1-42is added in co-cultured MI andAMSCsTo observe the cells morphology and test the variation of TNF-α、IL-6�'�NO byELLISAin each group after96hours.3. To build co-culture system of Transwell stratification: MI is cultured in upper Transwellroom, while neurons are cultured in six closet plate in down room. The group are dividedas follows:(1) Control group: Neurons are cultured in down room only, with medium in upperroom.(2) Activated by Aβ1-42group: Neurons are cultured in down room, with mediumcontainedAβ1-42in the concentration of10μM in upper room.(3) Activated by MI group: Neurons are cultured in down room, while the sameconcentration of MI are cultured in upper room.(4) Co-activated by Aβ1-42+MI group: Neurons are cultured in down room, while thesame concentration of MI are cultured in upper room with medium contained Aβ1-42in the concentration of10μM.(5) Co-cultured intervened by AMSCs group: Neurons are cultured in down room,while the same concentration of MI are cultured in upper room with medium containedAβ1-42in the concentration of10μM and the equal account ofAMSCs4. To observe the apoptosis rate of neurons under the immunofluorescence microscopyafter96hours in both Hoechst33342marked-method and AnnexinV-FITC/PImarked-method. Apoptosis rate=positive cells/total cells.5. Statistical analysis: all data are analyze by SPSS16.0software. P <0.05indicated astatistically significant difference.Result1. AMSCs which are primary cultured take an adherent growth after inoculation, have ahigh expression of CD90、CD105, low expression of CD45、HLA-DR by the identificationof flow Cytometry and have a positive expression of CD200.2. AMSCs can express neuron-specific markers MAP-2、GFAP after induction;3. MI which are divided cultured have a flat or ovoid cell body. The positive rate ofCD68are more than92%;Neurons which are primary cultured have a plentiful cell body with clear synaptic.Neuron-specific markers MAP-2can be seen both in cell body and synaptic;4. Test the variation of TNF-α、IL-6and NO by ELLISA(1) The content of TNF-α、IL-6and NO are improved secreted by MI under thestimulation ofAβ（P<0.05）;(2) The content of Inflammatory molecules such as TNF-α、IL-6and NO secreted byAMSCs co-cultured is lower than that of activated by Aβ1-42group and higher than that ofcontrol group（P<0.05）.5. Apoptosis rate of neurons tested byAnnexinV-FITC/PI method shows that:(1) Apoptosis rate of neurons is12.08in control group, while that co-cultured of MIandAMSCs is13.25, There is no significant difference（P>0.05）; (2) Apoptosis rate of co-cultured of Aβ and neurons is19.75. Compared with controlgroup, there is significant difference（P<0.01）;(3) Apoptosis rate of co-stimulated of Aβand MI increased to31.46compared withstimulated ofAβ, and this have statistical significance（P<0.01）;(4) Apoptosis rate of co-cultured added AMSCs can decrease to20.79, which ishigher than control group（P<0.01）, but lower than that of co-stimulated of Aβ and MIgroup.Conclusion1. AMSCs have rich source, amplification ability, low immunogenicity, with thecharacteristics of stem cells, and can be induced to differentiate into neuron-like cell;2. Aβ1-42can activate MI, promote its increased secretion of inflammatory cytokines,whileco-cultured AMSCs and MI can decrease the secretion of MI;3. Activation of MI mediated by Aβ1-42can induce neuronal apoptosis, but, AMSCs mayplay a protective role to neuronal by negatively regulating the activation of MI.