The Role Of Olfactory Ensheathing Cells On Astrocytes Activation And Injury

ObjectiveTo study the effect of olfactory ensheathing cells on reactive and injured astrocytes in vitro and investigate the relationship between the two cells. Methods(1) Culture olfactory ensheathing cells and astrocytes in vitro and identify the Purity(2) After purified astrocytes were passaged and grown to confluence.Some cultures were scratched to induce a reactive astrocyte phenotype. Injury model of astrocytes were prepared by H2O2 .(3) Olfactory ensheathing cells were added onto confluent astrocytes for 24 h. The differences of cell proliferation,expressions of GFAP and CSPG were measured among the normal group, active group,and the co-cultured group.(4) Astrocytes being injuried were co-culture with OEC culture medium for 72 h. Cell morphology as well as the number were observed by microscope; MTT was used to confect the proliferation; Flow cytometry (Annexin V/PI double-staining method) was used to detect cell apoptosis; ultrastructure changes were observed by transmission electron microscopy.Results(1) The proliferation of reactive astrocytes was increased. The expression of GFAP mRNA in reactive group was higher than that of normal group. The expression of GFAP and CSPG protein in reactive group were lower than these of normal group.(2) Compared with normal group,the cell number of injury model of astrocytes decreased significantly;Cell body of the survival shrinkage,cell processes shrinkage and broken obviously; The proliferation was decreased; Ultrastructure showed cell membrane rupture partialy, swollen mitochondria and endoplasmic reticulum, and the formation of cytoplasmic vacuolization.(3) After olfactory ensheathing cells were co-cultured with reactive astrocytes,the proliferation of reactive astrocytes was increased, a decrease in GFAP and CSPG expression was also observed(4) After being co-cultured with OEC culture medium, cell number of injury group increased; the survival cell morphology became normal; Ultrastructure showed better membrane integrity, normal morphology of endoplasmic reticulum, and mitochondrial swelling reduced, cytoplasmic vacuoles decreased significantly.ConclusionAfter being co-cultured with reactive astrocytes, olfactory ensheathing cells can promote the proliferation of reactive astrocytes,and reduce the astrocyte gliosis and expression of inhibitory cytokines; The astrocytes'necrosis and apoptosis can be reduced by OEC culture medium,this suggest that it can protect the injured astrocytes. At the same time ,OECCM can reduce the proliferation of survival injury astrocytes, reduce the astrocyte gliosis.