| Objectives:Alzheimer’s disease(AD)is a commonly neurodegenerative disease,which main pathological feature is the neuron toxic beta-amyloid(Aβ)deposits,neurofibrillary tangles(NFT)formed by excessive Tau protein,synaptic dysfunction.Major clinical symptoms include memory loss,cognitive impairment,deterioration of motor ability,currently there is no effective treatments.Human amniotic epithelial cells(hAECs)have characterizations of neurobiology and immunomodulatory,it provides the possibility of cell therapy for AD.Extraction of hAECs are limited,the number of cells needed for treatment cannot be met,usually need to be amplified in vitro.However,hAECs inevitably result in epithelial-mesenchymal transition(EMT)after serial passage amplification.It is not clear whether hAECsunderwent EMT would lose biological characteristics and would affect the biological treatment effect.This study observed thechanges of differentiation abilityand biological characteristics of hAECsunderwent EMT.Further,using the rat model of AD induced with Aβ25-35,we explored the therapic effect of hAECsunderwent EMT on AD model rats.Method:(1)hAECs were isolated from human amniotic tissues in full-term delivery by trypsin digestion,forcing it to undergo EMT by TGF-βin vitro,phenotypic identification by Flow cytometry(FCM)and Immunofluorescence(IF),and migration ability was observed by Wound Healing.(2)Rat model of AD was replicated by bilateral ventriclesinjection of Aβ25-35 in Wistarrats.The model rats were randomly divided into 4 groups:Model group,Mediumgroup,hAECstransplantationgroupandEMTphenotype hAECs(EMT-hAECs)transplantation group,with 12 rats in each group.Bilateral hippocampal in the cell transplantation group were injected with 10ul cell suspensionof hAECs or EMT-hAECs(5 x 10~5 cells),and the Medium group was injected with equivalent volume of DMEM/F12 medium without serum by the same route.TheNormal rats group was set as parallel control.After cell transplantation 2 weeks and 4 weeks,in each group,6animals were sacrificed,brain and peripheral blood were taken for pathological and immunological examination.(3)The changes of spatial discriminant learning and memory ability were observed by water maze test before and after cell transplantation.(4)Pathological changes in the hippocampus were observed by HE staining,and amyloid protein deposition were observed by sulfur S staining.(5)Immunohistochemical staining was used to observe the expression of CD68,Tau proteinand Aβ42,and its positive area integral optical density(IOD)value was analyzed by image-proplus 6.0 Image analysis system.(6)TUNEL assay was used to detect apoptosis in brain transplant areas.(7)The expression of Bax,Bcl-2 and Caspase-3 in brain tissue of each experimental group was detected by Immunofluorescence.(8)Changes of T-lymphocyte subsetscontaining in peripheral blood were detected by FCM.(9)Immunofluorescence staining was used to detect the survival and differentiation of hAECs and EMT-hAECs in the brain transplantation area.Results:(1)After 6 days of induction by TGF-β,hAECs showed mesenchymal cell like changes,the expression of epithelial cell marker CD326and E-cad were significantly down-regulated(all P<0.05),the interstitial cell marker N-cad was up-regulated,and the migration ability was enhanced,EMT-hAECs which obtained EMT phenotypecould differentiate into osteogenic,cartilage and adipocytes as well as hAECs,both of EMT-hAECs andhAECsexpressed neurofilament protein(NFP),A2B5,GFAP,CBX8.(2)Compared with normal rats,the escape incubation period increased significantly from the third day(P<0.01)and the number of crossing the platform decreased(P<0.05),indicating that AD modelsrats replicated by Aβ25-35had significantly spatial exploratory learning and memory disorder.Compared with the Model group and the Medium group,the escape incubation period of the hAECs and EMT-hAECs treatment groups were significantly shortened from the first day(all P<0.05),and the number of crossing the platform was significantly increased(all P<0.05),which was close to the Normal group(all P>0.05),while there was no significant difference between the two cell treatment groups(P>0.05).(3)Compared with the Model group and the Medium group,the hAECs and EMT-hAECs transplantation group showed a significant reduction in brain hippocampal lesion,neuronal apoptosis,and Aβ42 deposition and tau protein deposition(P<0.05),the expression of pro-apoptotic proteins Bax and Caspase-3 were up-regulated,while the expression of Bcl-2 apoptotic inhibitory protein was down-regulated,amyloid plaque were significantly reduced,and no significant difference was found between the two treatment groups.(4)Immunohistochemical staining showed that the number of CD68positive cells(microglia cells)in the hippocampus of the two treatment groups was significantly higher than of all the control groups(P>0.05).(5)Compared with the Model group and the Medium group,the percentage of peripheral blood Th1 and Th17 cells in the hAECs and EMT-hAECs treatment group decreased(P<0.05),while Th2 cells and Treg cells showed an up-regulated trend.(6)Immunofluorescence staining showed that hAECs and EMT-hAECswere still alive in the brain transplantation area of animals for 4 weeks after cell transplantationand expressed neuron-specific nucleoprotein(NeuN).Conclusions:This study provides the evidence that hAECsunderwent EMT may haveoriginalbiological characteristics and good biological therapeutic effect on AD model rats,suggesting that EMT-hAECs can possibly be applied for intracerebralallografting to treat neurologic diseases in which neurons are damaged. |
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