| Objective:To establish a method of obtainment,isolation and cultivation of Sprague Dawley(SD)rats olfactory mucosa mesenchymal stem cells(OM-MSCs)in vitro.To reserch the effect of OM-MSCs combined immunosuppressive therapy on the mice with alzheimer’s disease(AD).The levels of inflammatory factors,learning,memory ability and the formation of beta-amyloid were detected in peripheral blood through behavioral tests and immunohistochemical and enzyme-linked immunosorbent assay.The efficacy of OM-MSCs combined with immunosuppressive agents for the treatment of AD was detected.Methods:(1)Through the olfactory mucosa adherent method,olfactory mucosa were isolated,cultured in vitro between mesenchymal stem cells,and morphological observation,cell immunofluorescence technique is used to test mesenchymal stem cell and neural stem cell markers,flow cytometry to detect cell surface markers,to research the differentiation in the direction of bone tissue and adipose tissue,and cell cycle analysis.(2)AD rat model was established,and the experiment is divided into five group:control group,saline group,OM-MSCs group,immunosuppressant group and OM-MSCs + immunosuppressant group.After intervention,the average escape latency and the number of platforms of SD rats was detected and the improvement of learning and memory function was evaluated by using Morris water maze.The BrdU staining of OM-MSCs in the posterior brain was first detected by immunofluorescence technique.After the intervention,The degeneration and necrosis of the nerve cell in the hippocampal area of each group and Thioflavin fluorescence staining was carried out on the precipitation of A(3-amyloid protein in hippocampus region.Finally,the concentration of inflammatory cytokines(IL-6)and brain-derived neurotrophic factor(BDNF)in peripheral blood was detected by enzyme-linked immunosorbent assay.Results:(1)OM-MSCs were spindle cell-based,showing radial colony arrangement,and cells kept vigorous growth.Cell immunofluorescence showed OM-MSCs express two markers:STRO-1 and nestin.At the 4th passage,OM-MSCs were negative for CD19,CD31,CD34,CD45 and HLA-DR,but positive for CD90 and CD105.Following induction of osteoinduction or adipogenic induction,oil red O and alizarin red staining produced a strong positive.Cell cycle demonstrated that NM-MSCs were consistent with the growth characteristics of stem cells.(2)After 1 month of intervention in AD model rats,In the positioning navigation experiment,the immunosuppressive combined OM-MSCs group found the shortest incubation period compared with the other four groups.In the space exploration experiment,the combined group had more times than the other groups.BrdU staining of OM-MSCs can be detected in the hippocampus after 45 days intervention.HE staining showed that the cells in the combined group were the most orderly and denatured,and the necrotic cells decreased significantly.Sulfurin staining showed that The age spots formed by the precipitation of Aβ-amyloid protein in the pathological sections of the hippocampal area were significantly reduced than other four groups.After 45 days of intervention:The IL-6 concentration of OM-MSCs group was significantly reduced and the concentration of BDNF was significantly increased and the combined group had more significant results than the other four groups.Conclusion:(1)SD rats olfactory mucosa tissue adherence method is simple and can isolate and amplify OM-MSCs for animal experiment study in vitro.With multi-directional differentiation potential after induced in vitro,and has the general biological characteristics of mesenchymal stem cells.This experimental method has important practical significance to provide sufficient source of seed cells for tissue and cell engineering.(2)OM-MSCs can improve the learning and memory ability of AD rat model.The mechanism was related to the improvement of microenvironment in the lesion area after the migration of om-mscs to the intracerebral lesion area.It inhibits inflammatory response and the degeneration and necrosis of peripheral nerve cells through its own immune regulation and reduce the formation of Aβ-amyloid protein plaques in the hippocampus.(3)OM-MSCs combined immunosuppressive transplantation was more significant in the study and memory improvement of AD rats than OM-MSCs group.It shows that OM-MSCs has a synergistic effect with immunosuppressive agents. |
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