Role Of Pigment Epithelium Derived Factor In Hypoxic Injury Of Perivascular Resident Macrophages In Cochlea

Objective:To investigate the role of pigment epithelium derived factor（PEDF）in hypoxia injury of perivascular resident macrophages（PVMs）in mouse stria vascularis by establishing CoCl₂ model.Methods:Primary PVMs cell lines were obtained from stria vascularis explants of C57BL/6 mice aged 10-15 days in growth medium and purified by flow cytometry.The phenotype and genotype of PVMs was proved with the help of immunofluorescence staining and RT-PCR.CoCl₂was used to mimic anoxic environment,and the cell viability detected by CCK-8 was to determine the optimal concentration of CoCl₂ in simulated hypoxia environment.3-5 generations of primary cultured PVMs were used in the experiment.Enzyme linked immunosorbent assay（ELISA）was used to detect the content of PEDF.The effect of PEDF on apoptosis of PVMs in hypoxia environment was determined.Results:PVMs were selectively obtained from the stria vascularis of the cochlea of10-15 days old mice by cell culture medium and purified by flow cytometry.Phenotypic and morphological verification of PVMs:immunostaining showed F4/80（red）,phalloidin（green）and nucleus（blue）,PVMs had unique dendritic processes.Gene identification of PVMs in F4/80,GST and GSTα4 showed obvious bands in the molecular weight region of their respective PCR products.CCK-8 showed that the optimal concentration of CoCl₂ was 250μM.The results of ELISA showed that the content of PEDF in macrophage medium containing PVMs was increased,and the content of PEDF secreted by PVMs under hypoxia was higher than that under normoxic condition.Flow cytometry showed that apoptosis of PVMs could be seen in CoCl₂ simulated hypoxia environment.PEDF could reduce the apoptosis of PVMs in hypoxia environment.ConclusionHigh quality PVMs cells can be cultured from stria vascularis of cochlea by this method.PVMs cells secrete PEDF,and PEDF secretion increases under hypoxia environment.CoCl₂ can simulate the hypoxia environment in cells,and the optimal concentration is 250um.PEDF has protective effect on hypoxia injury induced by CoCl₂.