The Expression Of Nogo-A,NgR And RhoA In Oligodendrocyte Precursor Cells And The Changes After Oxygen & Glucose Deprivation In Vitro

Object: To obtain highly purified oligodendrocyte precursor lineage cells of newborn immature SD rats in vitro for the needs of further study.Method: The 2 day-old SD rats were sacrificed by disconnecting neck under aseptic conditions and the brain was taken out. The meninges and blood vessels on the brain were removed. The oligodendrocyte precursors of the brain were separated from astrocyte by orbital shaker and further purified by differential adhesion and finally cultured in chemically defined serum-free medium with appended N2, PDGF, bFGF. Immunofluorescence assay was applied to identify the separated cells with A2B5,O4 and O1 antibodies which represent different phases of differentiation of oligodendrocyte precursors.Result: Over 95ï¼…of cultured oligodendrocyte precursor cells were obtained. The oligodendrocyte progenitors are A2B5 and O4 positive, while immature oligodendrocytes are O4 and O1 positive.Conclusion: Separation and purification by shaking and differential adhesion and chemically defined medium are suitable and effective to obtain highly purified oligodendrocyte precursor cells. The output of cells increases notably and keep in immature phase by Object: To set up the model of oligodendrocyte precursors (OLPs) of immature SD rat deprived of oxygen & glucose and observe the changes of OLPs cells morphologically and in survival rate and apoptosis rate in different time of deletion of oxygen and glucose.Method: separate and culture highly purified oligodendrocyte precursor lineage cells and make identification of immature SD rat in vitro. Choose the cells that surface antibody A2B5 or O4 or O1 positive to culture in absence of oxygen and glucose conditions by using Na₂S₂O₄ and Earle's fluid in the medium for 10min, 30min, 60min and 90min, respectively to set up the oxygen & glucose deletion injured model(OGD) of OLPs . Meanwhile, the both of mature oligodendrocytes (OL) with deletion of oxygen & glucose and normal OLPs without deletion of oxygen & glucose are set up for the control groups. The morphologic changes of cells are observed by light microscope and electron microscope in different hypoxic duration and the livability of cells in each group is detected by MTT and the cell apoptosis rate is measured by DAPI dye. Results: (1) When Na₂S₂O₄'S concentration is 10mmol/L in culture medium, the oxygen pressure in culture medium without glucose keeps in very low or zero, so we choose 10mmol/L Na₂S₂O₄ as the property concentration for making the hypoxia model. (2) Compared with the two control groups, OLPs in OGD model was significantly damnified OLPs cells became markedly swelling and cell prominences reduced and the cell membrance ruptured. The nuclei were large and chromatin was condensed, and OLPs were collapsing and floating in culture medium. (3) MTT's results showed that OLPs livability rate in OGD model was significantly lower than those of the both control groups (P＜0. 05) . (4) The apoptosis rate by DAPI in OGD model group was significantly higher than those in the both control groups.Conclusion: The OGD model of oligodendrocyte precursors damage was successfully established, approving that hypoxic lesions of OLPs is maturity- dependent.Objective: To detect the expressions of Nogo-A, NgR & RhoA in OLPs and in OGD model in vitro to discuss their functions in restraining OLPs regeneration after the OLPs damage.Method: The OLPs were separated by improved separation and purification through agitation and then cultured in chemically defined medium. Set up OGD model of OLPs by N₂S₂O₄ in vitro. Immunofluorescence assay is applied to identify the separated cells with A2B5, O4, O1 antibodies and western blotting to observe the expressions ofNogo-A, NgR and RhoA in OLPs of different duration (10min, 30minand 60min, respectively)of OGD model and normal OLPs groups.Results: Nogo-A, NgR & RhoA were detected in purified OLPs and the positive signals of Nogo-A, NgR were located in the cell's body and the prominence whereas RhoA in cell plasm and prominence of OLPs. Compared with the control,the expressions of NogoA, increased significantly at 10min , 30min and 60min in OGD model(P＜0.05). The expressions of NgR increased significantly at 10min and 30min(P＜0.05),whereas decreased at 60min in OGD model(P＞0.05). The expressions of RhoA increased significantly at 10min ,30min and 60min of OGD compared with the control(P＜0.05).Conclusion: The expressions of Nogo-A and NgR proteins locate both in prominence and cell body of OLPs, and the expression of RhoA protein in the cell plasm and promincence of OLPs.After OGD injury, their expression all increased. Meanwhile, OLPs damage was consistant with the expression of Nogo-A,NgR and RhoA.suggesting that Nogo-A and NgR may play a role inOLPs damage, which might be due to the activation of RhoApassway. These findings may be useful for further experimental research in the understanding of pathogenesis ofrestraining OLPs' regeneration in premature periventricularleukomalacia (PVL).