| Alzheimer's disease (AD) is an incurable neurodegenerative disease with clinical manifestations that include a gradual decline in memory, cognitive function, personality, and linguistic function. One of the major pathological changes of AD is neurofibrillary tangles induced by hyperphosphorylated Tau protein in neurons. To date, traditional treatments for AD have little healing effect. With the development of stem cells, many types of stem cells have been used in AD therapy with some favorable effects, but the ideal stem cells still need be identified. Dental pulp stem cells (DPSCs) & Periodontal ligament stem cells (PDLSCs) are derived from neural crest cells and have a greater capacity for neurological differentiation compared with other mesoderm-derived stem cells. Thus, they achieve more beneficial effects in the treatment of neurological and central nervous system disorders. Currently, many in vivo and in vitro studies have shown that DPSCs & PDLSCs prevent and repair neuronal damage. However, there are few reports on the efficacy of human DPSCs & PDLSCs in the treatment of AD. In this study, we used okadaic acid (OA)-induced Tau phosphorylation in the human neuroblastoma cell line, SH-SY5Y, to establish an in vitro model of AD; and we further assessed a transwell co-culture system to evaluate the efficacy of DPSCs & PDLSCs treatment.The main methods and results of this study are presented as follows:1. The DPSCs, PDLSCs and UCMSCs were isolated by using explant or enzymatic methods, respectively. And using monoclonal method purified DPSCs and PDLSCs. Then observe and compare the differences of cell morphology, cell proliferation curves, surface markers expressions, tri-lineage differentiation potential by using inverted microscope, CCK-8 assay, flow cytometry, immunofluorescence staining, specifically staining and real-time PCR, respectively. Results showed cell morphology of DPSCs, PDLSCs and UCMSCs were slightly different, compared with UCMSCs, DPSCs and PDLSCs showed more elongated morphology. The cell proliferation curve of DPSCs and PDLSCs were significantly higher than UCMSCs. The three cell types were positively express the classical mesenchymal surface markers such as CD 166, CD 105, CD44, CD73 and CD90, but negative for CD34?CD31?CD45?CD3?HLA-DR. CD14?CD38; Immunofluorescence staining of the three cell types that indicated similar in expression of mesenchymal stem cell associated cell surface antigens such as vimentin and a-SMA,and the three cell types were able to differentiate into osteocytes, adipocytes and chondrocytes. Real-time PCR results indicated that no differences in the expression of OCN between hDPSCs and hUCMSCs, and more strongly than hPDLSCs after induced 2 weeks, however another osteogenic marker RUNX2 expression was increased in hDPSCs compared to hUCMSCs and hPDLSCs 2 weeks after osteogenic induction. Adipogenic differentiation marker PPAR-y was more strongly expressed in hDPSCs compared with hUCMSCs and hPDLSCs 3 weeks after adipogenic induction.2. To determine the best dosage of OA treatment in AD cell model, the diverse doses of OA were incubated with SH-SY5Y cells for 24h, and the changes in cell morphology and cell viability were observed. Cells in the model group were then incubated with 20 nmol/L OA for 24 h. This was followed by analyses of cell morphology, cell viability, apoptosis, cytoskeletal, microtubule structure and changes in Tau protein phosphorylation levels. Results showed 20nmol/L OA incubation with SH-SY5Y cells for 24 h can be taken as the proper conditions for developing the cell model of AD. 20nmol/L OA injury in SH-SY5Y cell lines, changed the cytomorphology of neurons, decreased cell viability, promoted the rate of apoptosis, and damaged the cytoskeletal structure. There was a significant difference with the control group. Western blot detection also showed, OA can enhance the Ser 396 phosphorylation of Tau protein levels.3. DPSCs & PDLSCs with AD model cells co-cultured 24h through Transwell or use their condition media cultured 12h, respectively. Results showed after treatment of DPSCs & PDLSCs, the cytomorphology of the OA-damaged SH-SY5Y cells was gradually restored with re-elongation of retracted dendrites. Staining by Cell Counting Kit-8 and Hoechst 33258 assays showed that DPSCs & PDLSCs increased the viability and relieved the apoptosis of SH-SY5Y cells. Dil labeling demonstrated that DPSCs & PDLSCs treated dendritic growth was healthy, with longer dendrites compared with OA-damaged cells. Additionally, specific staining for a-tubulin and F-actin demonstrated that DPSCs & PDLSCs treated cells had the morphology of restored neurons, with elongated dendrites, densely arranged microfilaments, and thickened microtubular fibrils. Ultrastructural analysis revealed that the cytoplasmic microtubules were relatively thick in DPSCs & PDLSCs treated cells, with normal quantities and areas of distribution. Results from western blot showed that DPSCs & PDLSCs suppressed OA-induced phosphorylation at Ser 396 of Tau protein.In summary, this study demonstrated that ? MSCs derived from different tissues have different biological characteristics, DPSCs,PDLSCs and UCMSCs have their own specific differentiation characteristics;? 20nmol/L OA-induced SH-SY5Y cell line 24h to establish AD-like cell model can be used for in vitro studies of AD;? This is the first report the therapeutic effect of DPSCs and PDLSCs on AD, the underlying mechanism may involve various hDPSC & hPDLSCs-secreted growth factors that antagonize phosphorylation of Tau protein. Results of this study showed that DPSCs and PDLSCs as odontogenic MSCs have the potential advantages in the treatment of AD, and provides an important scientific basis for using them in clinical therapy of AD. |
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