Effects Of Different Concentrations Of TNF-α On The GLAST Of Retinal Müller Cells

Objective: Rabbit retinal Müller cells are isolated and cultured in DMEM/F12with different concentrations of TNF-α. Through MTT assay, observe the growth andchange of retinal Müller cells.Through immunofluorescence technique, analyse thelocalization and semi-quantitative of glutamate/aspartate transporter (GLAST)in therabbit retinal Müller cells, to observe the effect of TNF-αon the capability of GLAST.Investigate Müller cells adjust the concentrations of glutamic acid of retina duringdifferent levels of TNF-α.Method: Using the enzyme-degisting method, the retina obtained from adultrabbit. Cultured cells were identified through the inverted phase contrast microscope,transmission electron microscope and immunofluorescence techniques. Through MTTassay, observe the vitality of Müller cells in the different concentrations of TNF-α（0pg/ml、20pg/ml、50pg/ml、100pg/ml、200pg/ml）. Using immunofluorescencetechniques to mark cells’ GLAST, measure its average light density by the imageanalysis software. Statistics analysis: datas were analysed by using the StatisticalPackage of the SPSS11.0. Discriptive data were given as mean±SD. Continuousvariable wre tested by analysis of one-way analysis of variance, The p-values of lessthan0.05being considered to be significant.Results:(1) Some adherent cells can be seen in the bottom after2-3days，cellmorphology has been basically stable around after9-10days and the entire bottlebottom was overspeads by cells after15days. Cultured cells’ cytoplasm were rich in8-10nm microfilaments. More than95%of cells were characterized byimmunofluorescence staining in glia fibrillary acidic protein and cellularretinaldehyde-binding protein.(2) Under the light microscope, by cell culture mediumof TNF-α concentration gradually elevated, cells refractive index increased, the partof cell processes increasingly retracted, the float-ing inactivation of cells graduallyincreased, and the adherent cells were partially dissolved. MTT assay cell viability, the OD values measured of the different concent-rations of TNF-α interventiongroups compared with the normal group are significant (p <0.05), and the OD valuedecreased gradually with the elevated concentration of TNF-α.(3) In the normal groupthe cytoplasm、 protrusions and membrane of the Müller cells showed greenfluorescent.The cell membrane and protrusions distributed visible granularfluorescence. In the intervention groups of different concentrations of TNF-αcompared with the normal group the GLAST expression on Müller cells weresignificantly reduced (p <0.05), and the GLAST expression decreases gradually withthe elevated concentration of TNF-α..Conclusion:(1) The retinal Müller cells cultured by the enzyme-degistingmethod, not only relatively high purity and the cycle is relatively short, can be usedfor experimental study;(2) TNF-α can inhibit the growth of Müller cells, and thestronger the inhibition the higher the concentration of TNF-α.;(3) GLAST is widelyexpressed in the retinal Müller cells, mainly on the cell membrane and processes.TNF-α can inhibit the activity of GLAST in Müller cells, and the stronger theinhibition the higher the concentration of TNF-α..