Experimental Study On The Effect Of ProNGF On Müller Cells And RGC Cells

ObjectiveTo Explore the effect of ProNGF on Müller cells and RGC cells,and to search for possible intracellular channels with increased TNFa expression.Methods1.Müller cells and RGC cells were cultured in vitro.Observe whether Sortilin and P75NTR are expressed in Müller cells and RGC cells by fluorescence microscopy.2.Müller cells and RGC cells were cultured in vitro,and different concentrations of ProNGF were added to the culture medium.Cell density was measured at 24h,48h,and 72h with enzyme-labeled instrument.3.After the Müller cells and RGC cells were cultured in high concentration ProNGF medium,the apoptotic trimers were observed by fluorescence microscopy.4.Müller cells and RGC cells were cultured in vitro,and a high concentration of ProNGF was added to the culture medium.After 24 hours,the supernatant was exchanged and the cell density was measured with an enzyme-labeled instrument for 24 hours.5.A rat optic nerve cut model was established to simulate the damaged conditions of retinal ganglion cell apoptosis.Observe the changes of retinal signal pathways in the retina at 1 day,4 days,1 week,2 weeks,and 4 weeks by proteomicsResults:1.Both RGC cells and Müller cells expressed Sortilin and P75NTR.2.After adding RGC cells and Müller cells with high concentration of ProNGF,the density of cells was not significantly reduced by enzyme-labeled instrument at 24h,48h,72h3.Müller cells and RGC cells were cultured with ProNGF.ProNGF,Sortilin,and P75NTR colocalized not only in the cytoplasm and nucleus,but also in the cell membrane.4.Add high concentration of ProNGF to RGC cells and Müller cell culture medium,exchange the supernatant for 24 hours,and then use an enzyme-labeled instrument to detect the apoptosis of RMC-1 at 24 hours.5.Signal pathway analysis was performed after the intersection of differential proteins at 5 time points after optic nerve cut in rats.The top 10 signal transduction pathways were found to be EIF2 Signaling,Phototransduction Pathway,Cardiac ?-adrenergic Signaling,Regulation of eIF4 and p70S6K Signaling,Regulation of eIF4 and p70S6K Signaling,Protein Kinase A Signaling,Purine Nucleotides De Novo Biosynthesis II,Huntington's Disease Signaling,mTOR Signaling,GP6 Signaling Pathway,Actin Cytoskeleton SignalingConclusion:In cell culture in vitro,both RGC cells and Müller cells expressed Sortilin and P75NTR,and ProNGF,Sortilin and P75NTR co-localized in the cytoplasm and nucleus,and on the cell membrane.Under the high concentration of ProNGF,neither Müller cells nor RGC cells apoptotic;after exchanging the supernatant,the cell density of RMC-1 was detected with a microplate reader after 24h,suggesting that RGC-5 may secrete cytokines after adding ProNGF Inhibited RMC-1 cell proliferation.The signal pathway analysis was performed after the intersection of differential proteins at 5 time points in the rat optic nerve cut model,and ten significant signal transduction pathway changes were found,indicating that after retinal ganglion cell apoptosis is damaged,activated pathways in the retina are very important.May be concentrated here.Figure [45] Table [22] Reference [47]...